U.S. patent application number 13/330902 was filed with the patent office on 2012-05-10 for mammalian cytokines; related reagents.
This patent application is currently assigned to Schering Corporation. Invention is credited to J. Fernando Bazan, Jeanne Cheung, Rene de Waal Malefyt, Robert A. Kastelein, Stefan Karl-Heinz Pflanz, Donna Rennick, Jacqueline C. Timans.
Application Number | 20120115164 13/330902 |
Document ID | / |
Family ID | 26668135 |
Filed Date | 2012-05-10 |
United States Patent
Application |
20120115164 |
Kind Code |
A1 |
Timans; Jacqueline C. ; et
al. |
May 10, 2012 |
MAMMALIAN CYTOKINES; RELATED REAGENTS
Abstract
Purified genes encoding a cytokine or composite cytokine from a
mammal, reagents related thereto including purified proteins,
specific antibodies, and nucleic acids encoding these molecules are
provided. Methods of using said reagents and diagnostic kits are
also provided.
Inventors: |
Timans; Jacqueline C.;
(Mountain View, CA) ; Pflanz; Stefan Karl-Heinz;
(Redwood City, CA) ; Kastelein; Robert A.;
(Portola Valley, CA) ; Bazan; J. Fernando; (Menlo
Park, CA) ; Rennick; Donna; (Los Altos, CA) ;
de Waal Malefyt; Rene; (Sunnyvale, CA) ; Cheung;
Jeanne; (Belmont, CA) |
Assignee: |
Schering Corporation
Kenilworth
NJ
|
Family ID: |
26668135 |
Appl. No.: |
13/330902 |
Filed: |
December 20, 2011 |
Related U.S. Patent Documents
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13025002 |
Feb 10, 2011 |
8080392 |
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13330902 |
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Mar 30, 2009 |
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13025002 |
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10777790 |
Feb 11, 2004 |
7579440 |
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10000776 |
Nov 30, 2001 |
7148330 |
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10777790 |
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Feb 22, 2001 |
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10000776 |
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Jul 30, 1999 |
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Current U.S.
Class: |
435/7.1 ;
435/320.1; 435/369; 435/375; 530/324; 530/325; 530/326; 530/387.9;
536/23.5; 536/24.31; 536/25.3 |
Current CPC
Class: |
C07K 14/54 20130101;
C12Q 1/6881 20130101; C07K 16/244 20130101; Y10S 436/808 20130101;
C07K 14/5434 20130101 |
Class at
Publication: |
435/7.1 ;
536/23.5; 435/320.1; 435/369; 530/326; 530/325; 530/324; 536/25.3;
536/24.31; 530/387.9; 435/375 |
International
Class: |
C12N 15/24 20060101
C12N015/24; C12N 5/073 20100101 C12N005/073; G01N 33/53 20060101
G01N033/53; C07K 14/54 20060101 C07K014/54; C07H 1/00 20060101
C07H001/00; C07K 16/24 20060101 C07K016/24; C12N 15/63 20060101
C12N015/63; C07K 7/08 20060101 C07K007/08 |
Claims
1. An isolated or recombinant polynucleotide encoding an antigenic
polypeptide comprising at least 17 contiguous amino acids from the
mature polypeptide from SEQ ID NO: 2, 4, 6, or 8.
2. The polynucleotide of claim 1, encoding a mature polypeptide
from SEQ ID NO: 2, 4, 6, or 8.
3. The polynucleotide of claim 1, which hybridizes at 55.degree.
C., less than 500 mM salt, and 50% formamide to the coding portions
of SEQ ID NO: 1, 3, 5, or 7.
4. The polynucleotide of claim 3, comprising at least 35 contiguous
nucleotides of the coding portion of SEQ ID NO: 1, 3, 5, or 7.
5. An expression vector comprising the polynucleotide of claim
1.
6. A host cell containing the expression vector of claim 5,
including a eukaryotic cell.
7. A method of making an antigenic polypeptide comprising
expressing a recombinant polynucleotide of claim 1.
8. A method for forming a duplex with a polynucleotide of claim 1,
2 5 comprising contacting said polynucleotide with a probe that
hybridizes, under stringent conditions, to at least 25 contiguous
nucleotides of the coding portion of SEQ ID NO: 1, 3, 5, or 7;
thereby forming said duplex.
9. A kit for the detection of a polynucleotide of claim 1,
comprising a polynucleotide that hybridizes, under stringent
hybridization conditions, to at least 17 contiguous nucleotides of
a polynucleotide of claim 1.
10. The kit of claim 9, wherein said probe is detectably
labeled.
11. A binding compound comprising an antibody binding site which
specifically binds to at least 17 contiguous amino acids from SEQ
ID NO: 2, 4, 6, or 8.
12. The binding compound of claim 11, wherein: a) said antibody
binding site is: 1) specifically immunoreactive with a polypeptide
of SEQ ID NO: 2, 4, 6 or 8; 2) raised against a purified or
recombinantly produced human IL-D80 or IL-27 protein; or 3) in a
monoclonal antibody, Fab, or F(ab)2; or b) said binding compound
is: 1) an antibody molecule; 2) a polyclonal antiserum; 3)
detectably labeled; 4) sterile; or 5) in a buffered
composition.
13. A method using the binding compound of claim 11, comprising
contacting said binding compound with a biological sample
comprising an antigen, wherein said contacting results in formation
of a binding compound:antigen complex.
14. The method of claim 13, wherein said biological sample is from
a human, and wherein said binding compound is an antibody.
15. A detection kit comprising said binding compound of claim 12,
and: a) instructional material for the use of said binding compound
for said detection; or b) a compartment providing segregation of
said binding compound.
16. A substantially pure or isolated antigenic polypeptide, which
binds to said binding composition of claim 11, and further
comprises at least 17 contiguous amino acids from SEQ ID NO: 2, 4,
6, or 8.
17. The polypeptide of claim 16, which: a) comprises at least a
fragment of at least 25 contiguous amino acid residues from a
primate IL-D80 or IL-27 protein; b) is a soluble polypeptide; c) is
detectably labeled; d) is in a sterile composition; e) is in a
buffered composition; f) binds to a cell surface receptor; g) is
recombinantly produced; or h) has a naturally occurring polypeptide
sequence.
18. The polypeptide of claim 17, which comprises at least 17
contiguous amino acids of SEQ ID NO: 2, 4, 6, or 8.
19. A method of modulating physiology or development of a cell or
tissue culture cells comprising contacting said cell with an
agonist or antagonist of a primate IL-D80 or IL-27.
20. The method of claim 19, wherein: a) said contacting is in
combination with an agonist or antagonist of IL-12; or b) said
contacting is with an antagonist, including binding composition
comprising an antibody binding site which specifically binds an
IL-D80 or IL-27.
21. A composite cytokine comprising a plurality of segments of SEQ
ID NO: 2, 4, 6, or 8 and SEQ ID NO: 10.
22. An isolated or recombinant polynucleotide encoding the
composite cytokine of claim 21.
23. A binding composition which specifically binds to an antigenic
fragment of the composite cytokine of claim 21.
24. A receptor subunit:ligand composition comprising a plurality of
polypeptide segments of SEQ ID NO: 2, 4, 6, or 8; SEQ ID NO:10; and
SEQ ID NO:12.
25. A binding composition which specifically binds to an antigenic
fragment of the receptor
Description
[0001] This filing is a continuation-in-part of US Utility patent
application of U.S. Ser. No. 09/791,497, filed Feb. 22, 2001, which
is a continuation-in-part of U.S. Ser. No. 09/568,699, filed Sep.
8, 2000, and claims benefit from U.S. Provisional Patent
Applications U.S. Ser. No, 60/146,581, filed Jul. 30, 1999; and
U.S. Ser. No. 60/147,763, filed Aug. 6, 1999, each of which are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention pertains to compositions related to
proteins which function in controlling biology and physiology of
mammalian cells, e.g., cells of a mammalian immune system. In
particular, it provides purified genes, proteins, antibodies, and
related reagents useful, e.g., to regulate activation, development,
differentiation, and function of various cell types, including
hematopoietic cells.
BACKGROUND OF THE INVENTION
[0003] Recombinant DNA technology refers generally to the technique
of integrating genetic information from a donor source into vectors
for subsequent processing, such as through introduction into a
host, whereby the transferred genetic information is copied and/or
expressed in the new environment. Commonly, the genetic information
exists in the form of complementary DNA (cDNA) derived from
messenger RNA (mRNA) coding for a desired protein product. The
carrier is frequently a plasmid having the capacity to incorporate
cDNA for later replication in a host and, in some cases, actually
to control expression of the cDNA and thereby direct synthesis of
the encoded product in the host.
[0004] For some time, it has been known that the mammalian immune
response is based on a series of complex cellular interactions,
called the "immune network". Recent research has provided new
insights into the inner workings of this network. While it remains
clear that much of the response does, in fact, revolve around the
network-like interactions of lymphocytes, macrophages,
granulocytes, and other cells, immunologists now generally hold the
opinion that soluble proteins, known as lymphokines, cytokines, or
monokines, play a critical role in controlling these cellular
interactions. Thus, there is considerable interest in the
isolation, characterization, and mechanisms of action of cell
modulatory factors, an understanding of which will lead to
significant advancements in the diagnosis and therapy of numerous
medical abnormalities, e.g., immune system disorders. Some of these
factors are hematopoietic growth and/or differentiation factors,
e.g., stem cell factor (SCF) or IL-12. See, e.g., Mire-Sluis and
Thorpe (1998) Cytokines Academic Press, San Diego; Thomson (ed.
1998) The Cytokine Handbook (3d ed.) Academic Press, San Diego;
Metcalf and Nicola (1995) The Hematopoietic Colony Stimulating
Factors Cambridge University Press; and Aggarwal and Gutterman
(1991) Human Cytokines Blackwell.
[0005] Lymphokines apparently mediate cellular activities in a
variety of ways. They have been shown to support the proliferation,
growth, and differentiation of pluripotential hematopoietic stem
cells into vast numbers of progenitors comprising diverse cellular
lineages making up a complex immune system. Proper and balanced
interactions between the cellular components are necessary for a
healthy immune response. The different cellular lineages often
respond in a different manner when lymphokines are administered in
conjunction with other agents.
[0006] Cell lineages especially important to the immune response
include two classes of lymphocytes: B-cells, which can produce and
secrete immunoglobulins (proteins with the capability of
recognizing and binding to foreign matter to effect its removal),
and T-cells of various subsets that secrete lymphokines and induce
or suppress the B-cells and various other cells (including other
T-cells) making up the immune network. These lymphocytes interact
with many other cell types.
[0007] Another important cell lineage is the mast cell (which has
not been positively identified in all mammalian species), which is
a granule-containing connective tissue cell located proximal to
capillaries throughout the body. These cells are found in
especially high concentrations in the lungs, skin, and
gastrointestinal and genitourinary tracts. Mast cells play a
central role in allergy-related disorders, particularly anaphylaxis
as follows: when selected antigens crosslink one class of
immunoglobulins bound to receptors on the mast cell surface, the
mast cell degranulates and releases mediators, e.g., histamine,
serotonin, heparin, and prostaglandins, which cause allergic
reactions, e.g., anaphylaxis.
[0008] IL-12 plays a critical role in cell-mediated immunity
(Gately et al. (1998); Trinchieri (1998); and Trinchieri (1995)).
Its activities are triggered through a high-affinity receptor
complex that gathers two closely related subunits, IL-12R.beta.1
and .beta.2 (Chua, et al. (1995); and Preskey et al. (1996b)). The
p35 subunit has been suggested to bind to a second a second soluble
cytokine receptor called EBI3 (Devergne et al. (1997)). As yet no
biological activity has been reported for the p35-EBI3 pair,
however, pairings of IL-12 subunits or IL-12-like subunits with
other cytokines may provide information about cell-mediated
immunity, e.g. T-cell regulation. Furthermore, the discovery of
receptors or receptor subunits for these heteromeric cytokines will
also provide information regarding immune regulation.
[0009] Research to better understand and treat various immune
disorders has been hampered by the general inability to maintain
cells of the immune system in vitro.
[0010] Immunologists have discovered that culturing these cells can
be accomplished through the use of T-cell and other cell
supernatants, which contain various growth factors, including many
of the lymphokines
[0011] From the foregoing, it is evident that the discovery and
development of new lymphokines and their related receptors or
receptor subunits e.g., related to the IL-6/IL-12 cytokine family
could contribute to new therapies for a wide range of degenerative
or abnormal conditions, which directly or indirectly involve the
immune system and/or hematopoietic cells. In particular, the
discovery and development of lymphokines which enhance or
potentiate the beneficial activities of known lymphokines would be
highly advantageous. The present invention provides new interleukin
compositions, receptor subunits, and related compounds, and methods
for their use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the comparison between SEQ ID NO: 2 and the
IL-D80 variant polypeptide of SEQ ID NO: 6.
[0013] FIG. 2 shows a comparison of rodent IL-D80 (SEQ ID NO: 4)
and variant rodent IL-D80 (SEQ ID NO: 8) polypeptide sequences.
[0014] FIG. 3 shows a comparison of human IL-D80 (SEQ ID NO: 6) and
rodent, e.g., mouse IL-D80 (SEQ ID NO: 8)
SUMMARY OF THE INVENTION
[0015] The present invention is directed to mammalian, e.g.,
rodent, canine, feline, primate, interleukin numbered DNAX 80
(IL-D80; p28) and its biological activities. The present invention
is also based upon the discovery of the association of IL-D80 with
the IL-12p40-like molecule, EBI3, and the binding of this composite
cytokine to an IL-12R.beta.2 subunit homologue known as WSX-1/TCCR.
The IL-D80/EBI3 composite cytokine is also known as IL-27. It
includes nucleic acids coding for polypeptides themselves and
methods for their production and use. The nucleic acids of the
invention are characterized, in part, by their homology to
complementary DNA (cDNA) sequences disclosed herein, and/or by
functional assays for growth factor- or cytokine-like activities,
e.g., IL-6/IL-12 family of cytokines (see Thomson (1998) The
Cytokine Handbook 3d ed., Academic Press, San Diego), applied to
the polypeptides, which are typically encoded by these nucleic
acids. Methods for modulating or intervening in the control of a
growth factor dependent physiology or an immune response are
provided.
[0016] The present invention is based, in part, upon the discovery
of new cytokine sequences exhibiting significant sequence and
structural similarity to the IL-6/1L12 family of cytokines In
particular, it provides primate, e.g., human, and rodent, e.g.,
mouse, sequences. Functional equivalents exhibiting significant
sequence homology will be available from other mammalian, e.g.,
cow, horse, and rat, mouse, and non-mammalian species.
[0017] In various protein embodiments, the invention provides: a
substantially pure or recombinant IL-D80 polypeptide exhibiting
identity over a length of at least about 12 amino acids to SEQ ID
NO: 2, 4, 6, or 8; a natural sequence IL-D80 of SEQ ID NO: 2, 4, 6,
or 8; and a fusion protein comprising IL-D80 sequence of SEQ ID NO:
2, 4, 6, or 8. In certain embodiments, the segment of identity is
at least about 14, 17, or 19 amino acids. In other embodiments, the
IL-D80 comprises a mature sequence comprising the sequences from
SEQ ID NO:2, 4, 6, or 8; or exhibits a post-translational
modification pattern distinct from natural IL-D80; or the
polypeptide: is from a warm blooded animal selected from a mammal,
including a primate; comprises at least one polypeptide segment of
SEQ ID NO: 2, 4, 6, or 8; exhibits a plurality of amino acid
residue fragments; is a natural allelic variant of IL-D80; has a
length at least about 30 amino acids; exhibits at least two
non-overlapping epitopes which are specific for a primate IL-D80;
exhibits sequence identity over a length of at least about 20 amino
acids to primate IL-D80; is glycosylated; has a molecular weight of
at least 10 kD with natural glycosylation; is a synthetic
polypeptide; is attached to a solid substrate; is conjugated to
another chemical moiety; is a 5-fold or less substitution from
natural sequence; or is a deletion or insertion variant from a
natural sequence. Preferred embodiments include a composition
comprising: a sterile IL-D80 polypeptide; or the IL-D80 polypeptide
and a carrier, wherein the carrier is: an aqueous compound,
including water, saline, and/or buffer; and/or formulated for oral,
rectal, nasal, topical, or parenteral administration. In fusion
protein embodiments, the protein can have: mature polypeptide
sequence from SEQ ID NO:2, 4, 6, or 8; a detection or purification
tag, including a FLAG, His6, or Ig sequence; and/or sequence of
another cytokine or chemokine, including an IL-12.
[0018] Kit embodiments include those with an IL-D80 polypeptide,
and: a compartment comprising the polypeptide; and/or instructions
for use or disposal of reagents in the kit.
[0019] In binding compound embodiments, the compound may have an
antigen binding site from an antibody, which specifically binds to
a natural IL-D80 polypeptide, wherein: the IL-D80 is a primate
protein; the binding compound is an Fv, Fab, or Fab2 fragment; the
binding compound is conjugated to another chemical moiety; or the
antibody: is raised against a peptide sequence of a mature
polypeptide portion from SEQ ID NO:2, 4, 6, or 8; is raised against
a mature IL-D80; is raised to a purified primate IL-D80; is
immunoselected; is a polyclonal antibody; binds to a denatured
IL-D80; exhibits a Kd of at least 30 .quadrature.M; is attached to
a solid substrate, including a bead or plastic membrane; is in a
sterile composition; or is detectably labeled, including a
radioactive or fluorescent label. Kits containing binding compounds
include those with: a compartment comprising the binding compound;
and/or instructions for use or disposal of reagents in the kit.
Often the kit is capable of making a qualitative or quantitative
analysis. Preferred compositions will comprise: a sterile binding
compound; or the binding compound and a carrier, wherein the
carrier is: an aqueous compound, including water, saline, and/or
buffer; and/or formulated for oral, rectal, nasal, topical, or
parenteral administration.
[0020] Nucleic acid embodiments include an isolated or recombinant
nucleic acid encoding an IL-D80 polypeptide or fusion protein,
wherein: the IL-D80 is from a primate; and/or the nucleic acid:
encodes an antigenic peptide sequence of SEQ ID NO:2, 4, 6, or 8;
encodes a plurality of antigenic peptide sequences of SEQ ID NO:2,
4, 6, or 8; exhibits identity to a natural cDNA encoding the
segment; is an expression vector; further comprises an origin of
replication; is from a natural source; comprises a detectable
label; comprises synthetic nucleotide sequence; is less than 6 kb,
preferably less than 3 kb; is from a primate, including a human;
comprises a natural full length coding sequence; is a hybridization
probe for a gene encoding the IL-D80; or is a PCR primer, PCR
product, or mutagenesis primer. The invention also provides a cell,
tissue, or organ comprising such a recombinant nucleic acid, and
preferably the cell will be: a prokaryotic cell; a eukaryotic cell;
a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a
mouse cell; a primate cell; or a human cell.
[0021] Kit embodiments include those with such nucleic acids, and:
a compartment comprising the nucleic acid; a compartment further
comprising the IL-D80 protein or polypeptide; and/or instructions
for use or disposal of reagents in the kit. Typically, the kit is
capable of making a qualitative or quantitative analysis.
[0022] In certain embodiments, the nucleic acid: hybridizes under
wash conditions of 30.degree. C. and less than 2M salt, or of
45.degree. C. and/or 500 mM salt, or 55.degree. C. and/or 150 mM
salt, to SEQ ID NO: 1, 3, 5, or 7; or exhibits identity over a
stretch of at least about 30, 55, or 75 nucleotides, to a primate
IL-D80.
[0023] The invention embraces a method of modulating physiology or
development of a cell or tissue culture cells comprising contacting
the cell with an agonist or antagonist of a primate IL-D80. The
method may be where: the contacting is in combination with an
agonist or antagonist of IL-12; or the contacting is with an
antagonist, including a binding composition comprising an antibody
binding site which specifically binds an IL-D80.
[0024] The invention further provides a composite cytokine (IL-27)
comprising a plurality of segments of SEQ ID NO:2, 4, 6, or 8 and
SEQ ID NO:10. Also encompassed is an isolated or recombinant
polynucleotide encoding the composite cytokine of said composite
cytokine Further provided is a receptor subunit:ligand composition
comprising a plurality of polypeptide segments of SEQ ID NO:2, 4,
6, or 8, SEQ ID NO:10, and SEQ ID NO:12.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] All references cited herein are incorporated herein by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference.
[0026] I. General
[0027] The present invention provides amino acid sequences and DNA
sequences encoding various mammalian proteins, which are cytokines,
e.g., which are secreted molecules which can mediate a signal
between immune or other cells. See, e.g., Paul (1997) Fundamental
Immunology (3d ed.) Raven Press, N.Y. The full length cytokines,
and fragments, or antagonists will be useful in physiological
modulation of cells expressing a receptor. It is likely that IL-D80
or IL-27 has either stimulatory or inhibitory effects on
hematopoietic cells, including, e.g., lymphoid cells, such as
T-cells, B-cells, natural killer (NK) cells, macrophages, dendritic
cells, hematopoietic progenitors, etc. In particular, the IL-27
composite cytokine may play a role in inflammation, including, but
not limited to ulcerative colitis, arthritis, etc. The proteins
will also be useful as antigens, e.g., immunogens, for raising
antibodies to various epitopes on the protein, both linear and
conformational epitopes.
[0028] A cDNA encoding IL-D80 was identified from various primate,
e.g., human, sequences of BACs of Chromosome 16. See, e.g.,
CIT987SK-A-575C2, and CIT987SK-A-761H5. The molecule was designated
huIL-D80. A human EST has been identified and described, human EST
AI085007. A mouse EST AA266872 has also been identified and
described.
[0029] The primate, e.g., human, gene will encode a small soluble
cytokine-like protein, of about 216 amino acids (for SEQ ID NO: 2)
or about 243 amino acids (for SEQ ID NO: 6). See SEQ. ID. NOs: 1,
2, 5, and 6. Exon boundaries are likely to correspond to about
219/220; 393/394; 492/493; and 551/552 of SEQ ID NO:1. Coding
segments corresponding to those boundaries are particularly
interesting. Translated amino acid sequence, which is encoded by
nucleotides 193 to 918 of SEQ ID NO:1, is shown in SEQ ID NO:
2.
[0030] A predicted signal cleavage site may exist between about
residues 25-30 of SEQ ID NO: 2; helix A is predicted to run from
about residues 33-38 to about residues 54-59 of SEQ ID NO: 2; helix
B is predicted to run from about residues 85-90 to about residues
111-116 of SEQ ID NO: 2; helix C is predicted to run from about
residues 121-126 to about residues 154-159 of SEQ ID NO: 2; and
helix D is predicted to run from about residues 201-206 to about
residues 228-233 of SEQ ID NO: 2.
[0031] SEQ ID NO: 5 shows a variant of IL-D80 and SEQ ID NO: 6 is
the encoded polypeptide. FIG. 1 shows the comparison between SEQ ID
NO: 2 and the IL-D80 variant polypeptide of SEQ ID NO: 6.
Structural motifs are as indicated above with the appropriate
change in residue positions.
[0032] The corresponding rodent polynucleotide sequence of IL-D80
is shown in SEQ ID NO: 3. Exon boundaries are likely to run from
about 198/199; 360/361; 459/460; and 618/619. The predicted
polypeptide sequence, which runs from about nucleotide 199 to 891
of SEQ ID NO: 3, is shown in SEQ ID NO: 4. The predicted signal
cleavage site runs from about residue 16-21 of SEQ ID NO: 4; helix
A is predicted to run from about residue 21-26 to about residue
41-46; helix B is predicted to run from about residue 72-77 to
about residue 101-106; helix C is predicted to run from about
residue 108-133 to about residue 141-146; and helix D is predicted
to run from about residue 185-190 to about residue 211-215. All
postions refer to SEQ ID NO: 4. A variant rodent IL-D80
polynucleotide sequence is shown in SEQ ID NO: 7 and the predicted
polypeptide sequence is shown in SEQ ID NO: 6. A comparison of
rodent IL-D80 (SEQ ID NO: 4) and variant rodent IL-D80 (SEQ ID NO:
8) polypeptide sequences is shown in FIG. 2.
[0033] IL-D80 exhibits structural motifs characteristic of a member
of the long chain cytokines belonging to the IL-6/IL-12 family of
cytokines The structural homology of IL-D80 to related cytokine
proteins suggests related function of this molecule.
[0034] The IL-D80 cDNA sequences mature proteins with calculated
molecular mass of 24.5 and 23.6 kDa. No N-glycosylation sites are
found in hIL-D80, but several O-glycosylation sites are predicted.
Murine IL-D80 contains one potential N-glycosylation site (N85).
Transient expression of mp28 in the presence or absence of
tunicamycin indicated that mp28 is indeed N-linked glycosylated.
Both human and mouse IL-D80 display an unusual sequence insertion
in the predicted loop region between helix C and D. In hIL-D80, the
C-D loop contains a stretch of 13 glutamic acid residues; mp28
displays 14 negatively charged residues in this region, interrupted
by one lysine residue. This highly charged sequence has not been
observed in any other helical cytokine and most likely will affect
the biophysical properties of the protein in solution. Overall,
human and mouse IL-D80 are 74% identical.
[0035] Comparison of the sequences will also provide an
evolutionary tree. This can be generated, e.g., using the TreeView
program in combination with the ClustalX analysis software program.
See Thompson, et al. Nuc. Acids Res. 25:4876-4882; and TreeView,
Page, IBLS, University of Glasgow, e-mail rpage@bio.gla.ac.uk;
http://taxonomy.zoology.gla.ac.uk.rod.treeview.html.
[0036] Co-transfection of human Epstein-Barr virus-induced gene 3
(EBI3; GenBank NM005755; Devergne, et al. (1996) J. Virol.
70:1143-1153; SEQ ID NOs: 9 and 10) cDNA and human IL-D80 cDNA
leads to enhanced secretion of IL-D80. IL-D80 co-immunoprecipitated
with EBI3, and conversely, EBI3 co-immunoprecipitated with IL-D80.
This indicates that these two proteins form a composite factor that
either itself has biological functions (that neither protein has on
its own) or EBI3 is used as a shuttle to release IL-D80 in the
supernatant. Of note, EBI3 is also expressed in vivo by activated
antigen presenting cells (APCs) and at very high levels by
placental syncytiotrophoblasts. The present invention provides the
first evidence that the IL-80D/EBI3 composite cytokine (IL-27)
binds to an IL-12R-like subunit, WSX-1/TCCR (See, e.g., GenBank
AF265242; Chen, et al. (2000) Nature 407:916-920; SEQ ID NO: 11 and
12).
[0037] Biologically, IL-27 is produced by antigen presenting cells
(APCs). In contrast to other similar heterodimers made by APCs,
i.e., IL-12 (p35+p40) and IL-23 (p19+p40), kinetic analysis of
IL-27 showed that this composite cytokine is produced earlier in
activation of APCs. Thus, IL-27 can be a potent adjuvant of a Th1
response.
[0038] The primary activity of IL-27 triggers rapid clonal
expansion of antigen specific naive human and mouse CD4+ T cells.
Moreover, it promotes Th1 polarization and IFN.gamma. production of
naive CD4+ T cells. Mechanistically, these naive T cells are primed
to response to IL-27 by the production of this composite cytokine
by the APCs which interact with these cells. These activities of
IL-27 are dependent on simultaneous T cell receptor activation and
occur in synergy with IL-12.
[0039] IL-D80 or IL-27 agonists, or antagonists, may also act as
functional or receptor antagonists. Thus, IL-D80, IL-27,
WSX-1/TCCR, or its antagonists, may be useful in the treatment of
abnormal medical conditions, including immune disorders, e.g., T
cell immune deficiencies, inflammation, or tissue rejection, or in
cardiovascular or neurophysiological conditions.
[0040] The natural antigens are capable of mediating various
biochemical responses which lead to biological or physiological
responses in target cells. The preferred embodiment characterized
herein is from human, but other primate, or other species
counterparts exist in nature. Additional sequences for proteins in
other mammalian species, e.g., primates, canines, felines, and
rodents, should also be available, particularly the domestic animal
species. See below. The descriptions below are directed, for
exemplary purposes, to a human IL-D80 or IL-27, but are likewise
applicable to related embodiments from other species.
[0041] II. Purified IL-D80 or IL-27
[0042] Mammlian IL-D80 amino acid sequence, is shown in several
embodiments, e.g., SEQ ID NO: 2, 4, 6, or 8. EBI3 amino acid
sequence is provided in SEQ ID NO: 10. Other naturally occurring
nucleic acids which encode the protein can be isolated by standard
procedures using the provided sequence, e.g., PCR techniques, or by
hybridization. These amino acid sequences, provided amino to
carboxy, are important in providing sequence information for the
cytokine allowing for distinguishing the protein antigen from other
proteins and exemplifying numerous variants. Moreover, the peptide
sequences allow preparation of peptides to generate antibodies to
recognize such segments, and nucleotide sequences allow preparation
of oligonucleotide probes, both of which are strategies for
detection or isolation, e.g., cloning, of genes encoding such
sequences.
[0043] As used herein, the term "human soluble IL-D80 or IL-27"
shall encompass, when used in a protein context, a protein having
amino acid sequence corresponding to a soluble polypeptide shown in
SEQ ID NO: 2 or 6, or significant fragments thereof. Preferred
embodiments comprise a plurality of distinct, e.g., nonoverlapping,
segments of the specified length. Typically, the plurality will be
at least two, more usually at least three, and preferably 5, 7, or
even more. While the length minima are provided, longer lengths, of
various sizes, may be appropriate, e.g., one of length 7, and two
of length 12.
[0044] Binding components, e.g., antibodies, typically bind to an
IL-D80 or IL-27 with high affinity, e.g., at least about 100 nM,
usually better than about 30 nM, preferably better than about 10
nM, and more preferably at better than about 3 nM. Counterpart
proteins will be found in mammalian species other than human, e.g.,
other primates, ungulates, or rodents. Non-mammalian species should
also possess structurally or functionally related genes and
proteins, e.g., birds or amphibians.
[0045] The term "polypeptide" as used herein includes a significant
fragment or segment, and encompasses a stretch of amino acid
residues of at least about 8 amino acids, generally at least about
12 amino acids, typically at least about 16 amino acids, preferably
at least about 20 amino acids, and, in particularly preferred
embodiments, at least about 30 or more amino acids, e.g., 35, 40,
45, 50, 60, 75, 100, etc. Such fragments may have ends which begin
and/or end at virtually all positions, e.g., beginning at residues
1, 2, 3, etc., and ending at, e.g., 150, 149, 148, etc., in all
practical combinations. Particularly interesting peptides have ends
corresponding to structural domain boundaries, e.g., helices A, B,
C, and/or D.
[0046] The term "binding composition" refers to molecules that bind
with specificity to IL-D80 or IL-27, e.g., in an antibody-antigen
interaction. The specificity may be more or less inclusive, e.g.,
specific to a particular embodiment, or to groups of related
embodiments, e.g., primate, rodent, etc. It also includes
compounds, e.g., proteins, which specifically associate with IL-D80
or IL-27, including in a natural physiologically relevant
protein-protein interaction, either covalent or non-covalent. The
molecule may be a polymer, or chemical reagent. A functional analog
may be a protein with structural modifications, or it may be a
molecule which has a molecular shape which interacts with the
appropriate binding determinants. The compounds may serve as
agonists or antagonists of a receptor binding interaction, see,
e.g., Goodman, et al. (eds.) Goodman & Gilman's: The
Pharmacological Bases of Therapeutics (current ed.) Pergamon
Press.
[0047] Substantially pure, e.g., in a protein context, typically
means that the protein is free from other contaminating proteins,
nucleic acids, or other biologicals derived from the original
source organism. Purity may be assayed by standard methods,
typically by weight, and will ordinarily be at least about 40%
pure, generally at least about 50% pure, often at least about 60%
pure, typically at least about 80% pure, preferably at least about
90% pure, and in most preferred embodiments, at least about 95%
pure. Carriers or excipients will often be added.
[0048] Solubility of a polypeptide or fragment depends upon the
environment and the polypeptide. Many parameters affect polypeptide
solubility, including temperature, electrolyte environment, size
and molecular characteristics of the polypeptide, and nature of the
solvent. Typically, the temperature at which the polypeptide is
used ranges from about 4.degree. C. to about 65.degree. C. Usually
the temperature at use is greater than about 18.degree. C. For
diagnostic purposes, the temperature will usually be about room
temperature or warmer, but less than the denaturation temperature
of components in the assay. For therapeutic purposes, the
temperature will usually be body temperature, typically about
37.degree. C. for humans and mice, though under certain situations
the temperature may be raised or lowered in situ or in vitro.
[0049] The size and structure of the polypeptide should generally
be in a substantially stable state, and usually not in a denatured
state. The polypeptide may be associated with other polypeptides in
a quaternary structure, e.g., to confer solubility, or associated
with lipids or detergents.
[0050] The solvent and electrolytes will usually be a biologically
compatible buffer, of a type used for preservation of biological
activities, and will usually approximate a physiological aqueous
solvent. Usually the solvent will have a neutral pH, typically
between about 5 and 10, and preferably about 7.5. On some
occasions, one or more detergents will be added, typically a mild
non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS
(3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), or a
low enough concentration as to avoid significant disruption of
structural or physiological properties of the protein. In other
instances, a harsh detergent may be used to effect significant
denaturation.
[0051] The above will also be applicable to the IL-D80 or
IL-27/EBI3 composite cytokine, where SEQ ID NO: 10 is the
polypeptide sequence of EBI3.
[0052] III. Physical Variants
[0053] This invention also encompasses proteins or peptides having
substantial amino acid sequence identity with the amino acid
sequence of the IL-D80 or IL-27 antigen. The variants include
species, polymorphic, or allelic variants.
[0054] Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. See also Needleham, et al. (1970) J.
Mol. Biol. 48:443-453; Sankoff, et al. (1983) Chapter One in Time
Warps, String Edits, and Macromolecules: The Theory and Practice of
Sequence Comparison, Addison-Wesley, Reading, Mass.; and software
packages from IntelliGenetics, Mountain View, Calif.; and the
University of Wisconsin Genetics Computer Group, Madison, Wis.
Sequence identity changes when considering conservative
substitutions as matches. Conservative substitutions typically
include substitutions within the following groups: glycine,
alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. The conservation may apply to biological
features, functional features, or structural features. Homologous
amino acid sequences are typically intended to include natural
polymorphic or allelic and interspecies variations of a protein
sequence. Typical homologous proteins or peptides will have from
25-100% identity (if gaps can be introduced), to 50-100% identity
(if conservative substitutions are included) with the amino acid
sequence of the IL-D80 or IL-27. Identity measures will be at least
about 35%, generally at least about 40%, often at least about 50%,
typically at least about 60%, usually at least about 70%,
preferably at least about 80%, and more preferably at least about
90%.
[0055] The isolated IL-D80 or IL-27 DNA can be readily modified by
nucleotide substitutions, nucleotide deletions, nucleotide
insertions, and inversions of short nucleotide stretches. These
modifications result in novel DNA sequences which encode these
antigens, their derivatives, or proteins having similar
physiological, immunogenic, antigenic, or other functional
activity. These modified sequences can be used to produce mutant
antigens or to enhance expression. Enhanced expression may involve
gene amplification, increased transcription, increased translation,
and other mechanisms. "Mutant IL-D80 or IL-27" encompasses a
polypeptide otherwise falling within the sequence identity
definition of the IL-D80 or IL-27 as set forth above, but having an
amino acid sequence which differs from that of IL-D80 or IL-27 as
normally found in nature, whether by way of deletion, substitution,
or insertion. This generally includes proteins having significant
identity with a protein having sequence of SEQ ID NO: 2, 4, 6, or
8, or the foregoing in association with SEQ ID NO: 10 and as
sharing various biological activities, e.g., antigenic or
immunogenic, with those sequences, and in preferred embodiments
contain most of the natural full length disclosed sequences. Full
length sequences will typically be preferred, though truncated
versions will also be useful, likewise, genes or proteins found
from natural sources are typically most desired. Similar concepts
apply to different IL-D80 or IL-27 proteins, particularly those
found in various warm blooded animals, e.g., mammals and birds.
These descriptions are generally meant to encompass many IL-D80 or
IL-27 proteins, not limited to the particular mammalian embodiments
specifically discussed.
[0056] IL-D80 or IL-27 mutagenesis can also be conducted by making
amino acid insertions or deletions. Substitutions, deletions,
insertions, or any combinations may be generated to arrive at a
final construct. Insertions include amino- or carboxy- terminal
fusions. Random mutagenesis can be conducted at a target codon and
the expressed mutants can then be screened for the desired
activity. Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are well known
in the art, e.g., by M13 primer mutagenesis or polymerase chain
reaction (PCR) techniques. See, e.g., Sambrook, et al. (1989);
Ausubel, et al. (1987 and Supplements); and Kunkel, et al. (1987)
Methods in Enzymol. 154:367-382. Preferred embodiments include,
e.g., 1-fold, 2-fold, 3-fold, 5-fold, 7-fold, etc., preferably
conservative substitutions at the nucleotide or amino acid levels.
Preferably the substitutions will be away from the conserved
cysteines, and often will be in the regions away from the helical
structural domains. Such variants may be useful to produce specific
antibodies, and often will share many or all biological
properties.
[0057] The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
proteins. A heterologous fusion protein is a fusion of proteins or
segments which are naturally not normally fused in the same manner.
A similar concept applies to heterologous nucleic acid
sequences.
[0058] In addition, new constructs may be made from combining
similar functional domains from other proteins. For example,
target-binding or other segments may be "swapped" between different
new fusion polypeptides or fragments. See, e.g., Cunningham, et al.
(1989) Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol.
Chem. 263:15985-15992.
[0059] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence, e.g., PCR techniques.
[0060] Structural analysis can be applied to this gene, in
comparison to the IL-12 family of cytokines In particular,
.beta.-sheet and .alpha.-helix residues can be determined using,
e.g., RASMOL program, see Bazan, et al. (1996) Nature 379:591;
Lodi, et al. (1994) Science 263:1762-1766; Sayle and Milner-White
(1995) TIBS 20:374-376; and Gronenberg, et al. (1991) Protein
Engineering 4:263-269. Preferred residues for substitutions include
the surface exposed residues which would be predicted to interact
with receptor. Other residues which should conserve function will
be conservative substitutions, particularly at position far from
the surface exposed residues.
[0061] The above will also be applicable for the IL-D80 or IL-27
(i.e., IL-D80 +EBI3) composite cytokine where SEQ ID NO: 10 is the
polypeptide sequence of EBI3.
[0062] IV. Functional Variants
[0063] The blocking of physiological response to IL-D80 or the
IL-27 composite cytokine may result from the competitive inhibition
of binding of the ligand to its receptor.
[0064] In vitro assays of the present invention will often use
isolated protein, soluble fragments comprising receptor binding
segments of these proteins, or fragments attached to solid phase
substrates. These assays will also allow for the diagnostic
determination of the effects of either binding segment mutations
and modifications, or cytokine mutations and modifications, e.g.,
IL-D80 or IL-27 analogs.
[0065] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to the
cytokine, or receptor binding fragments compete with a test
compound.
[0066] "Derivatives" of IL-D80 or IL-27 antigens include amino acid
sequence mutants from naturally occurring forms, glycosylation
variants, and covalent or aggregate conjugates with other chemical
moieties. Covalent derivatives can be prepared by linkage of
functionalities to groups which are found in IL-D80 or IL-27 amino
acid side chains or at the N- or C- termini, e.g., by standard
means. See, e.g., Lundblad and Noyes (1988) Chemical Reagents for
Protein Modification, vols. 1-2, CRC Press, Inc., Boca Raton, Fla.;
Hugli (ed. 1989) Techniques in Protein Chemistry, Academic Press,
San Diego, Calif.; and Wong (1991) Chemistry of Protein Conjugation
and Cross Linking, CRC Press, Boca Raton, Fla.
[0067] In particular, glycosylation alterations are included, e.g.,
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing, or in further processing
steps. See, e.g., Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also
embraced are versions of the peptides with the same primary amino
acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine.
[0068] Fusion polypeptides between IL-D80 or IL-27 and other
homologous or heterologous proteins are also provided. Many
cytokine receptors or other surface proteins are multimeric, e.g.,
homodimeric entities, and a repeat construct may have various
advantages, including lessened susceptibility to proteolytic
cleavage. Typical examples are fusions of a reporter polypeptide,
e.g., luciferase, with a segment or domain of a protein, e.g., a
receptor-binding segment, so that the presence or location of the
fused ligand may be easily determined. See, e.g., Dull, et al.,
U.S. Pat. No. 4,859,609. Other gene fusion partners include
bacterial .beta.-galactosidase, trpE, Protein A, .beta.-lactamase,
alpha amylase, alcohol dehydrogenase, yeast alpha mating factor,
and detection or purification tags such as a FLAG sequence of His6
sequence. See, e.g., Godowski, et al. (1988) Science
241:812-816.
[0069] Fusion peptides will typically be made by either recombinant
nucleic acid methods or by synthetic polypeptide methods.
Techniques for nucleic acid manipulation and expression are
described generally, e.g., in Sambrook, et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed.), vols. 1-3, Cold Spring
Harbor Laboratory; and Ausubel, et al. (eds. 1993) Current
Protocols in Molecular Biology, Greene and Wiley, NY. Techniques
for synthesis of polypeptides are described, e.g., in Merrifield
(1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986) Science
232: 341-347; Atherton, et al. (1989) Solid Phase Peptide
Synthesis: A Practical Approach, IRL Press, Oxford; and Grant
(1992) Synthetic Peptides: A User's Guide, W.H. Freeman, NY.
Refolding methods may be applicable to synthetic proteins.
[0070] This invention also contemplates the use of derivatives of
IL-D80 or IL-27 proteins other than variations in amino acid
sequence or glycosylation. Such derivatives may involve covalent or
aggregative association with chemical moieties or protein carriers.
Covalent or aggregative derivatives will be useful as immunogens,
as reagents in immunoassays, or in purification methods such as for
affinity purification of binding partners, e.g., other antigens. An
IL-D80 or IL-27 can be immobilized by covalent bonding to a solid
support such as cyanogen bromide-activated SEPHAROSE, by methods
which are well known in the art, or adsorbed onto polyolefin
surfaces, with or without glutaraldehyde cross-linking, for use in
the assay or purification of anti-IL-D80 or IL-27 antibodies or an
alternative binding composition. The IL-D80 or IL-27 proteins can
also be labeled with a detectable group, e.g., for use in
diagnostic assays. Purification of IL-D80 or IL-27 may be effected
by an immobilized antibody or complementary binding partner, e.g.,
binding portion of a receptor.
[0071] A solubilized IL-D80 or IL-27, or fragments of this
invention can be used as an immunogen for the production of
antisera or antibodies specific for binding. Purified antigen can
be used to screen monoclonal antibodies or antigen-binding
fragments, encompassing antigen binding fragments of natural
antibodies, e.g., Fab, Fab', F(ab).sub.2, etc. Purified IL-D80 or
IL-27 antigens can also be used as a reagent to detect antibodies
generated in response to the presence of elevated levels of the
cytokine, which may be diagnostic of an abnormal or specific
physiological or disease condition. This invention contemplates
antibodies raised against amino acid sequences encoded by
nucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7, or fragments
of proteins containing it. Also contemplated are sequences encoding
the IL-D80 or IL-27 cytokines, or fragments thereof. In particular,
this invention contemplates antibodies having binding affinity to
or being raised against specific domains, e.g., helices A, B, C, or
D.
[0072] The present invention contemplates the isolation of
additional closely related species variants. Southern and Northern
blot analysis will establish that similar genetic entities exist in
other mammals. It is likely that IL-D80 or IL-27s are widespread in
species variants, e.g., rodents, lagomorphs, carnivores,
artiodactyla, perissodactyla, and primates.
[0073] The invention also provides means to isolate a group of
related antigens displaying both distinctness and similarities in
structure, expression, and function. Elucidation of many of the
physiological effects of the molecules will be greatly accelerated
by the isolation and characterization of additional distinct
species or polymorphic variants of them. In particular, the present
invention provides useful probes for identifying additional
homologous genetic entities in different species.
[0074] The isolated genes will allow transformation of cells
lacking expression of an IL-D80 or IL-27, e.g., either species
types or cells which lack corresponding proteins and exhibit
negative background activity. This should allow analysis of the
function of IL-D80 or IL-27 in comparison to untransformed control
cells.
[0075] Dissection of critical structural elements which effect the
various physiological functions mediated through these antigens is
possible using standard techniques of modern molecular biology,
particularly in comparing members of the related class. See, e.g.,
the homolog-scanning mutagenesis technique described in Cunningham,
et al. (1989) Science 243:1339-1336; and approaches used in O'Dowd,
et al. (1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et
al. (1990) EMBO J. 9:4381-4390.
[0076] Intracellular functions would probably involve receptor
signaling. However, protein internalization may occur under certain
circumstances, and interaction between intracellular components and
cytokine may occur. Specific segments of interaction of IL-D80 or
IL-27 with interacting components may be identified by mutagenesis
or direct biochemical means, e.g., cross-linking or affinity
methods. Structural analysis by crystallographic or other physical
methods will also be applicable. Further investigation of the
mechanism of signal transduction will include study of associated
components which may be isolatable by affinity methods or by
genetic means, e.g., complementation analysis of mutants.
[0077] Further study of the expression and control of IL-D80 or
IL-27 will be pursued. The controlling elements associated with the
antigens should exhibit differential physiological, developmental,
tissue specific, or other expression patterns. Upstream or
downstream genetic regions, e.g., control elements, are of
interest.
[0078] Structural studies of the IL-D80 or IL-27 antigens will lead
to design of new antigens, particularly analogs exhibiting agonist
or antagonist properties on the molecule. This can be combined with
previously described screening methods to isolate antigens
exhibiting desired spectra of activities.
[0079] V. Antibodies
[0080] Antibodies can be raised to various epitopes of the IL-D80
or IL-27 proteins, including species, polymorphic, or allelic
variants, and fragments thereof, both in their naturally occurring
forms and in their recombinant forms. Additionally, antibodies can
be raised to IL-D80 or IL-27s in either their active forms or in
their inactive forms, including native or denatured versions.
Anti-idiotypic antibodies are also contemplated.
[0081] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the antigens can be
raised by immunization of animals with conjugates of the fragments
with immunogenic proteins. Monoclonal antibodies are prepared from
cells secreting the desired antibody. These antibodies can be
screened for binding to normal or defective IL-D80 or IL-27s, or
screened for agonistic or antagonistic activity, e.g., mediated
through a receptor. Antibodies may be agonistic or antagonistic,
e.g., by sterically blocking binding to a receptor. These
monoclonal antibodies will usually bind with at least a K.sub.D of
about 1 mM, more usually at least about 300 .mu.M, typically at
least about 100 .mu.M, more typically at least about 30 .mu.M,
preferably at least about 10 .mu.M, and more preferably at least
about 3 .mu.M or better.
[0082] An IL-D80 or IL-27 protein that specifically binds to or
that is specifically immunoreactive with an antibody generated
against a defined immunogen, such as an immunogen consisting of the
amino acid sequence of SEQ ID NO: 2, 4, 6, or 8, or any of the
foregoing in association with SEQ ID NO: 10, is typically
determined in an immunoassay. The immunoassay typically uses a
polyclonal antiserum which was raised, e.g., to a polypeptide of
SEQ ID NO: 2, 4, 6, or 8, or any of the foregoing in association
with SEQ ID NO: 10. This antiserum is selected to have low
crossreactivity against other IL12 family members, e.g., human or
rodent IL-12, preferably from the same species, and any such
crossreactivity is removed by immunoabsorption prior to use in the
immunoassay.
[0083] In order to produce antisera for use in an immunoassay, the
protein of SEQ ID NO: 2, 4, 6, or 8, or the foregoing in
association with SEQ ID NO: 10, or a combination thereof, is
isolated as described herein. For example, recombinant protein may
be produced in a mammalian cell line. An appropriate host, e.g., an
inbred strain of mice such as Balb/c, is immunized with the
selected protein, typically using a standard adjuvant, such as
Freund's adjuvant, and a standard mouse immunization protocol (see
Harlow and Lane, supra). Alternatively, a synthetic peptide derived
from the sequences disclosed herein and conjugated to a carrier
protein can be used an immunogen. Polyclonal sera are collected and
titered against the immunogen protein in an immunoassay, e.g., a
solid phase immunoassay with the immunogen immobilized on a solid
support. Polyclonal antisera with a titer of 10.sup.4 or greater
are selected and tested for their cross reactivity against other
IL-12 family members, e.g., rodent IL-12, using a competitive
binding immunoassay such as the one described in Harlow and Lane,
supra, at pages 570-573. Preferably at least one other IL-12 family
member is used in this determination in conjunction with, e.g., the
primate IL-12. The IL-12 family members can be produced as
recombinant proteins and isolated using standard molecular biology
and protein chemistry techniques as described herein.
[0084] Immunoassays in the competitive binding format can be used
for the crossreactivity determinations. For example, the protein of
SEQ ID NO: 2 or 6 can be immobilized to a solid support. Proteins
added to the assay compete with the binding of the antisera to the
immobilized antigen. The ability of the above proteins to compete
with the binding of the antisera to the immobilized protein is
compared to the protein of SEQ ID NO: 2 or 6. Similarly, the
composite cytokine of SEQ ID NO: 2 or 6 in association with SEQ ID
NO: 10 can be used. The crossreactivity for the above proteins is
calculated, using standard calculations. Those antisera with less
than 10% crossreactivity with each of the proteins listed above are
selected and pooled. The cross-reacting antibodies are then removed
from the pooled antisera by immunoabsorption with the above-listed
proteins.
[0085] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein to the immunogen protein (e.g., the IL-12 like
protein of SEQ ID NO: 2, 4, 6, or 8, or any of the foregoing in
association with SEQ ID NO: 10). In order to make this comparison,
the two proteins are each assayed at a wide range of concentrations
and the amount of each protein required to inhibit 50% of the
binding of the antisera to the immobilized protein is determined.
If the amount of the second protein required is less than twice the
amount of the protein of the selected protein or proteins that is
required, then the second protein is said to specifically bind to
an antibody generated to the immunogen.
[0086] The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing antibodies,
they can be screened for ability to bind to the antigens without
inhibiting binding to a receptor. As neutralizing antibodies, they
can be useful in competitive binding assays. They will also be
useful in detecting or quantifying IL-D80 or IL-27 protein or its
receptors, e.g., WSX-1/TCCR (SEQ ID NO: 12). See, e.g., Chan (ed.
1987) Immunology: A Practical Guide, Academic Press, Orlando, Fla.;
Price and Newman (eds. 1991) Principles and Practice of
Immunoassay, Stockton Press, N.Y.; and Ngo (ed. 1988) Nonisotopic
Immunoassay, Plenum Press, N.Y. Cross absorptions, depletions, or
other means will provide preparations of defined selectivity, e.g.,
unique or shared species specificities. These may be the basis for
tests which will identify various groups of antigens.
[0087] Further, the antibodies, including antigen binding
fragments, of this invention can be potent antagonists that bind to
the antigen and inhibit functional binding, e.g., to a receptor
which may elicit a biological response. They also can be useful as
non-neutralizing antibodies and can be coupled to toxins or
radionuclides so that when the antibody binds to antigen, a cell
expressing it, e.g., on its surface, is killed. Further, these
antibodies can be conjugated to drugs or other therapeutic agents,
either directly or indirectly by means of a linker, and may effect
drug targeting.
[0088] Antigen fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. An antigen and its fragments
may be fused or covalently linked to a variety of immunogens, such
as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid,
etc. See Microbiology, Hoeber Medical Division, Harper and Row,
1969; Landsteiner (1962) Specificity of Serological Reactions,
Dover Publications, New York; Williams, et al. (1967) Methods in
Immunology and Immunochemistry, vol. 1, Academic Press, New York;
and Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH
Press, NY, for descriptions of methods of preparing polyclonal
antisera.
[0089] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)
Basic and Clinical Immunology (4th ed.), Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.), Academic Press, New York; and particularly in Kohler and
Milstein (1975) in Nature 256:495-497, which discusses one method
of generating monoclonal antibodies.
[0090] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar vectors.
See, Huse, et al. (1989) "Generation of a Large Combinatorial
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-546. The
polypeptides and antibodies of the present invention may be used
with or without modification, including chimeric or humanized
antibodies. Frequently, the polypeptides and antibodies will be
labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents, teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567; Moore, et al., U.S. Pat. No. 4,642,334; and Queen, et
al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.
[0091] The antibodies of this invention can also be used for
affinity chromatography in isolating the protein. Columns can be
prepared where the antibodies are linked to a solid support. See,
e.g., Wilchek et al. (1984) Meth. Enzymol. 104:3-55. The converse
may be used to purify antibodies.
[0092] Antibodies raised against each IL-D80 or IL-27 will also be
useful to raise anti-idiotypic antibodies. These will be useful in
detecting or diagnosing various immunological conditions related to
expression of the respective antigens.
[0093] VI. Nucleic Acids
[0094] The described peptide sequences and the related reagents are
useful in detecting, isolating, or identifying a DNA clone encoding
IL-D80 or IL-27, e.g., from a natural source. Typically, it will be
useful in isolating a gene from mammal, and similar procedures will
be applied to isolate genes from other species, e.g., warm blooded
animals, such as birds and mammals. Cross hybridization will allow
isolation of IL-D80 or IL-27 from the same, e.g., polymorphic
variants, or other species. A number of different approaches will
be available to successfully isolate a suitable nucleic acid
clone.
[0095] The purified protein or defined peptides are useful for
generating antibodies by standard methods, as described above.
Synthetic peptides or purified protein can be presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane (1989) Antibodies: A Laboratory Manual, Cold
Spring Harbor Press.
[0096] For example, the specific binding composition could be used
for screening of an expression library made from a cell line which
expresses an IL-D80 or IL-27. Screening of intracellular expression
can be performed by various staining or immunofluorescence
procedures. Binding compositions could be used to affinity purify
or sort out cells expressing a surface fusion protein.
[0097] The peptide segments can also be used to predict appropriate
oligonucleotides to screen a library. The genetic code can be used
to select appropriate oligonucleotides useful as probes for
screening. See, e.g., SEQ ID NO: 1, 3, 5, or 7, or any of the
foregoing in addition to SEQ ID NO: 9. In combination with
polymerase chain reaction (PCR) techniques, synthetic
oligonucleotides will be useful in selecting correct clones from a
library. Complementary sequences will also be used as probes,
primers, or antisense strands. Various fragments should be
particularly useful, e.g., coupled with anchored vector or poly-A
complementary PCR techniques or with complementary DNA of other
peptides.
[0098] This invention contemplates use of isolated DNA or fragments
to encode an antigenic or biologically active corresponding IL-D80
or IL-27 polypeptide, particularly lacking the portion coding the
untranslated 5' portion of the described sequence. In addition,
this invention covers isolated or recombinant DNA which encodes a
biologically active protein or polypeptide and which is capable of
hybridizing under appropriate conditions with the DNA sequences
described herein. Said biologically active protein or polypeptide
can be an intact antigen, or fragment, and have an amino acid
sequence disclosed in, e.g., SEQ ID NO: 2, 4, 6, or 8, or any of
the foregoing in association with SEQ ID NO: 10, particularly a
mature, secreted polypeptide. Further, this invention covers the
use of isolated or recombinant DNA, or fragments thereof, which
encode proteins which exhibit high identity to a secreted IL-D80 or
IL-27. The isolated DNA can have the respective regulatory
sequences in the 5' and 3' flanks, e.g., promoters, enhancers,
poly-A addition signals, and others. Alternatively, expression may
be effected by operably linking a coding segment to a heterologous
promoter, e.g., by inserting a promoter upstream from an endogenous
gene.
[0099] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other components which naturally accompany a native sequence, e.g.,
ribosomes, polymerases, and/or flanking genomic sequences from the
originating species. The term embraces a nucleic acid sequence
which has been removed from its naturally occurring environment,
and includes recombinant or cloned DNA isolates and chemically
synthesized analogs or analogs biologically synthesized by
heterologous systems. A substantially pure molecule includes
isolated forms of the molecule. Generally, the nucleic acid will be
in a vector or fragment less than about 50 kb, usually less than
about 30 kb, typically less than about 10 kb, and preferably less
than about 6 kb.
[0100] An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
minor heterogeneity. This heterogeneity is typically found at the
polymer ends or portions not critical to a desired biological
function or activity.
[0101] A "recombinant" nucleic acid is defined either by its method
of production or its structure. In reference to its method of
production, e.g., a product made by a process, the process is use
of recombinant nucleic acid techniques, e.g., involving human
intervention in the nucleotide sequence, typically selection or
production. Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments which are
not naturally contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring mutants. Thus, e.g.,
products made by transforming cells with any unnaturally occurring
vector is encompassed, as are nucleic acids comprising sequence
derived using any synthetic oligonucleotide process. Such is often
done to replace a codon with a redundant codon encoding the same or
a conservative amino acid, while typically introducing or removing
a sequence recognition site.
[0102] Alternatively, it is performed to join together nucleic acid
segments of desired functions to generate a single genetic entity
comprising a desired combination of functions not found in the
commonly available natural forms. Restriction enzyme recognition
sites are often the target of such artificial manipulations, but
other site specific targets, e.g., promoters, DNA replication
sites, regulation sequences, control sequences, or other useful
features may be incorporated by design. A similar concept is
intended for a recombinant, e.g., fusion, polypeptide. Specifically
included are synthetic nucleic acids which, by genetic code
redundancy, encode polypeptides similar to fragments of these
antigens, and fusions of sequences from various different species
or polymorphic variants.
[0103] A significant "fragment" in a nucleic acid context is a
contiguous segment of at least about 17 nucleotides, generally at
least about 22 nucleotides, ordinarily at least about 29
nucleotides, more often at least about 35 nucleotides, typically at
least about 41 nucleotides, usually at least about 47 nucleotides,
preferably at least about 55 nucleotides, and in particularly
preferred embodiments will be at least about 60 or more
nucleotides, e.g., 67, 73, 81, 89, 95, 150, 200, 250, 300, 500,
etc.
[0104] A DNA which codes for an IL-D80 or IL-27 protein will be
particularly useful to identify genes, mRNA, and cDNA species which
code for related or similar proteins, as well as DNAs which code
for homologous proteins from different species. There will be
homologs in other species, including primates, rodents, canines,
felines, birds, and fish. Various IL-D80 or IL-27 proteins should
be homologous and are encompassed herein. However, even proteins
that have a more distant evolutionary relationship to the antigen
can readily be isolated under appropriate conditions using these
sequences if they are sufficiently homologous. Primate IL-D80 or
IL-27 proteins are of particular interest.
[0105] Recombinant clones derived from the genomic sequences, e.g.,
containing introns, will be useful for transgenic studies,
including, e.g., transgenic cells and organisms, and for gene
therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt
(ed.) Encyclopedia of Immunology, Academic Press, San Diego, pp.
1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al. (1991)
Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson
(ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, IRL Press, Oxford; Rosenberg (1992) J. Clinical Oncology
10:180-199; and Cournoyer and Caskey (1993) Ann. Rev. Immunol.
11:297-329. Alternatively, expression may be effected by operably
linking a coding segment to a heterologous promoter, e.g., by
inserting a promoter upstream from an endogenous gene. See, e.g.,
Treco, et al. WO96/29411 or U.S. Ser. No. 08/406,030.
[0106] Substantial homology, e.g., identity, in the nucleic acid
sequence comparison context means either that the segments, or
their complementary strands, when compared, are identical when
optimally aligned, with appropriate nucleotide insertions or
deletions, in at least about 50% of the nucleotides, generally at
least about 58%, ordinarily at least about 65%, often at least
about 71%, typically at least about 77%, usually at least about
85%, preferably at least about 95 to 98% or more, and in particular
embodiments, as high as about 99% or more of the nucleotides.
Alternatively, substantial homology exists when the segments will
hybridize under selective hybridization conditions, to a strand, or
its complement, typically using a sequence of IL-D80 or IL-27,
e.g., in SEQ ID NO: 1, 3, 5, or 7, or any of the foregoing in
association with SEQ ID NO: 9. Typically, selective hybridization
will occur when there is at least about 55% identity over a stretch
of at least about 30 nucleotides, preferably at least about 75%
over a stretch of about 25 nucleotides, and most preferably at
least about 90% over about 20 nucleotides. See, Kanehisa (1984)
Nuc. Acids Res. 12:203-213. The length of identity comparison, as
described, may be over longer stretches, and in certain embodiments
will be over a stretch of at least about 17 nucleotides, usually at
least about 28 nucleotides, typically at least about 40
nucleotides, and preferably at least about 75 to 100 or more
nucleotides.
[0107] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions. Stringent
temperature conditions will usually include temperatures in excess
of about 30.degree. C., usually in excess of about 37.degree. C.,
typically in excess of about 55.degree. C., 60.degree. C., or
65.degree. C., and preferably in excess of about 70.degree. C.
Stringent salt conditions will ordinarily be less than about 1000
mM, usually less than about 400 mM, typically less than about 250
mM, preferably less than about 150 mM, including about 100, 50, or
even 20 mM. However, the combination of parameters is much more
important than the measure of any single parameter. See, e.g.,
Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370. Hybridization
under stringent conditions should give a background of at least
2-fold over background, preferably at least 3-5 or more.
[0108] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0109] Optical alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by visual inspection (see generally Ausubel
et al., supra).
[0110] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
sequence identity. It also plots a tree or dendrogram showing the
clustering relationships used to create the alignment. PILEUP uses
a simplification of the progressive alignment method of Feng and
Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is
similar to the method described by Higgins and Sharp (1989) CABIOS
5:151-153. The program can align up to 300 sequences, each of a
maximum length of 5,000 nucleotides or amino acids. The multiple
alignment procedure begins with the pairwise alignment of the two
most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. For example, a reference sequence can be
compared to other test sequences to determine the sequence identity
relationship using the following parameters: default gap weight
(3.00), default gap length weight (0.10), and weighted end
gaps.
[0111] Another example of algorithm that is suitable for
determining sequence identity and sequence similarity is the BLAST
algorithm, which is described Altschul, et al. (1990) J. Mol. Biol.
215:403-410. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(http:www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul, et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Extension of the
word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLAST program uses as defaults a
wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and
Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915) alignments (B)
of 50, expectation (E) of 10, M=5, N=4, and a comparison of both
strands.
[0112] In addition to calculating sequence identity, the BLAST
algorithm also performs a statistical analysis of the similarity
between two sequences (see, e.g., Karlin and Altschul (1993) Proc.
Nat'l Acad. Sci. USA 90:5873-5787). One measure of similarity
provided by the BLAST algorithm is the smallest sum probability
(P(N)), which provides an indication of the probability by which a
match between two nucleotide or amino acid sequences would occur by
chance. For example, a nucleic acid is considered similar to a
reference sequence if the smallest sum probability in a comparison
of the test nucleic acid to the reference nucleic acid is less than
about 0.1, more preferably less than about 0.01, and most
preferably less than about 0.001.
[0113] A further indication that two nucleic acid sequences of
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid, as
described below. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules hybridize to each other under
stringent conditions, as described below.
[0114] IL-D80 or IL-27 from other mammalian species can be cloned
and isolated by cross-species hybridization of closely related
species. Homology may be relatively low between distantly related
species, and thus hybridization of relatively closely related
species is advisable. Alternatively, preparation of an antibody
preparation which exhibits less species specificity may be useful
in expression cloning approaches.
[0115] VII. Making IL-D80 or IL-27; Mimetics
[0116] DNA which encodes the IL-D80 or IL-27 or fragments thereof
can be obtained by chemical synthesis, screening cDNA libraries, or
screening genomic libraries prepared from a wide variety of cell
lines or tissue samples. See, e.g., Okayama and Berg (1982) i Mol.
Cell. Biol. 2:161-170; Gubler and Hoffman (1983) Gene 25:263-269;
and Glover (ed. 1984) DNA Cloning: A Practical Approach, IRL Press,
Oxford. Alternatively, the sequences provided herein provide useful
PCR primers or allow synthetic or other preparation of suitable
genes encoding an IL-D80 or IL-27; including naturally occurring
embodiments.
[0117] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length IL-D80 or IL-27 or fragments
which can in turn, e.g., be used to generate polyclonal or
monoclonal antibodies; for binding studies; for construction and
expression of modified molecules; and for structure/function
studies. There may be a need for a chaparone protein for efficient
secretion, or additional steps may be necessary to retrieve the
protein from the intracellular compartment.
[0118] Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles which
enable the integration of DNA fragments into the genome of the
host. See, e.g., Pouwels, et al. (1985 and Supplements) Cloning
Vectors: A Laboratory Manual, Elsevier, N.Y.; and Rodriguez, et al.
(eds. 1988) Vectors: A Survey of Molecular Cloning Vectors and
Their Uses, Buttersworth, Boston, Mass.
[0119] For purposes of this invention, DNA sequences are operably
linked when they are functionally related to each other. For
example, DNA for a presequence or secretory leader is operably
linked to a polypeptide if it is expressed as a preprotein or
participates in directing the polypeptide to the cell membrane or
in secretion of the polypeptide. A promoter is operably linked to a
coding sequence if it controls the transcription of the
polypeptide; a ribosome binding site is operably linked to a coding
sequence if it is positioned to permit translation. Usually,
operably linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences that in
turn control expression. See, e.g., Rodriguez, et al., Chapter 10,
pp. 205-236; Balbas and Bolivar (1990) Methods in Enzymology
185:14-37; and Ausubel, et al. (1993) Current Protocols in
Molecular Biology, Greene and Wiley, NY.
[0120] Representative examples of suitable expression vectors
include pCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.
5:1136-1142; pMClneo Poly-A, see Thomas, et al. (1987) Cell
51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
See, e.g., Miller (1988) Ann. Rev. Microbiol. 42:177-199.
[0121] It will often be desired to express an IL-D80 or IL-27
polypeptide in a system which provides a specific or defined
glycosylation pattern. See, e.g., Luckow and Summers (1988)
Bio/Technology 6:47-55; and Kaufman (1990) Meth. Enzymol.
185:487-511.
[0122] The IL-D80 or IL-27, or a fragment thereof, may be
engineered to be phosphatidyl inositol (PI) linked to a cell
membrane, but can be removed from membranes by treatment with a
phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol
phospholipase-C. This releases the antigen in a biologically active
form, and allows purification by standard procedures of protein
chemistry. See, e.g., Low (1989) Biochim. Biophys. Acta
988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner,
et al. (1991) J. Cell Biol. 114:1275-1283.
[0123] Now that the IL-D80 or IL-27 has been characterized,
fragments or derivatives thereof can be prepared by conventional
processes for synthesizing peptides. These include processes such
as are described in Stewart and Young (1984) Solid Phase Peptide
Synthesis, Pierce Chemical Co., Rockford, Ill.; Bodanszky and
Bodanszky (1984) The Practice of Peptide Synthesis,
Springer-Verlag, New York; Bodanszky (1984) The Principles of
Peptide Synthesis, Springer-Verlag, New York; and Villafranca (ed.
1991) Techniques in Protein Chemistry II, Academic Press, San
Diego, Calif.
[0124] VIII. Uses
[0125] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
IL-D80 or IL-27 mediated conditions, or below in the description of
kits for diagnosis. The gene may be useful in forensic sciences,
e.g., to distinguish rodent from human, or as a marker to
distinguish between different cells exhibiting differential
expression or modification patterns.
[0126] This invention also provides reagents with significant
commercial and/or therapeutic potential. The IL-D80 or IL-27
(naturally occurring or recombinant), fragments thereof, and
antibodies thereto, along with compounds identified as having
binding affinity to IL-D80 or IL-27, should be useful as reagents
for teaching techniques of molecular biology, immunology, or
physiology. Appropriate kits may be prepared with the reagents,
e.g., in practical laboratory exercises in production or use of
proteins, antibodies, cloning methods, histology, etc.
[0127] The reagents will also be useful in the treatment of
conditions associated with abnormal physiology or development,
including inflammatory conditions. They may be useful in vitro
tests for presence or absence of interacting components, which may
correlate with success of particular treatment strategies. In
particular, modulation of physiology of various, e.g.,
hematopoietic or lymphoid, cells will be achieved by appropriate
methods for treatment using the compositions provided herein. See,
e.g., Thomson (ed. 1998) The Cytokine Handbook (3d ed.) Academic
Press, San Diego; Metcalf and Nicola (1995) The Hematopoietic
Colony Stimulating Factors Cambridge University Press; and Aggarwal
and Gutterman (1991) Human Cytokines Blackwell Pub.
[0128] For example, a disease or disorder associated with abnormal
expression or abnormal signaling by an IL-D80 or IL-27 should be a
likely target for an agonist or antagonist. Similarly, the binding
partner of the IL-27 composite cytokine, WSX-1/TCCR, should also be
a target. The new cytokine should play a role in regulation or
development of hematopoietic cells, e.g., lymphoid cells, which
affect immunological responses, e.g., inflammation and/or
autoimmune disorders. Alternatively, it may affect vascular
physiology or development, or neuronal effects.
[0129] In particular, the cytokine should mediate, in various
contexts, cytokine synthesis by the cells, proliferation, etc.
Antagonists of IL-D80 or IL-27, such as mutein variants of a
naturally occurring form of IL-D80 or IL-27 or blocking antibodies,
may provide a selective and powerful way to block immune responses,
e.g., in situations as inflammatory or autoimmune responses. See
also Samter, et al. (eds.) Immunological Diseases vols. 1 and 2,
Little, Brown and Co.
[0130] Various abnormal conditions are known in different cell
types which will produce IL-D80 or IL-27, e.g., as evaluated by
mRNA expression by Northern blot analysis. See Berkow (ed.) The
Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway,
N.J.; Thorn, et al. Harrison's Principles of Internal Medicine,
McGraw-Hill, N.Y.; and Weatherall, et al. (eds.) Oxford Textbook of
Medicine, Oxford University Press, Oxford. Many other medical
conditions and diseases involve activation by macrophages or
monocytes, and many of these will be responsive to treatment by an
agonist or antagonist provided herein. See, e.g., Stites and Ten
(eds.; 1991) Basic and Clinical Immunology Appleton and Lange,
Norwalk, Conn.; and Samter, et al. (eds.) Immunological Diseases
Little, Brown and Co. These problems should be susceptible to
prevention or treatment using compositions provided herein.
[0131] IL-D80 or IL-27, antagonists, antibodies, etc., can be
purified and then administered to a patient, veterinary or human.
These reagents can be combined for therapeutic use with additional
active or inert ingredients, e.g., in conventional pharmaceutically
acceptable carriers or diluents, e.g., immunogenic adjuvants, along
with physiologically innocuous stabilizers, excipients, or
preservatives. These combinations can be sterile filtered and
placed into dosage forms as by lyophilization in dosage vials or
storage in stabilized aqueous preparations. This invention also
contemplates use of antibodies or binding fragments thereof,
including forms which are not complement binding.
[0132] Drug screening using IL-D80, IL-27, WSX-1/TCCR or fragments
thereof can be performed to identify compounds having binding
affinity to or other relevant biological effects on IL-D80 or IL-27
functions, including isolation of associated components. Subsequent
biological assays can then be utilized to determine if the compound
has intrinsic stimulating activity and is therefore a blocker or
antagonist in that it blocks the activity of the cytokine Likewise,
a compound having intrinsic stimulating activity can activate the
signal pathway and is thus an agonist in that it simulates the
activity of IL-D80 or IL-27. This invention further contemplates
the therapeutic use of blocking antibodies to IL-D80, IL-27, or
WSX-1/TCCR as antagonists and of stimulatory antibodies as
agonists. This approach should be particularly useful with other
IL-D80 or IL-27 species variants.
[0133] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, physiological state of the patient,
and other medicants administered. Thus, treatment dosages should be
titrated to optimize safety and efficacy. Typically, dosages used
in vitro may provide useful guidance in the amounts useful for in
situ administration of these reagents. Animal testing of effective
doses for treatment of particular disorders will provide further
predictive indication of human dosage. Various considerations are
described, e.g., in Gilman, et al. (eds.) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, latest Ed., Pergamon Press;
and Remington's Pharmaceutical Sciences, latest ed., Mack
Publishing Co., Easton, Pa. Methods for administration are
discussed therein and below, e.g., for oral, intravenous,
intraperitoneal, or intramuscular administration, transdermal
diffusion, and others. Pharmaceutically acceptable carriers will
include water, saline, buffers, and other compounds described,
e.g., in the Merck Index, Merck & Co., Rahway, N.J. Dosage
ranges would ordinarily be expected to be in amounts lower than 1
mM concentrations, typically less than about 10 .mu.M
concentrations, usually less than about 100 nM, preferably less
than about 10 .mu.M (picomolar), and most preferably less than
about 1 fM (femtomolar), with an appropriate carrier. Slow release
formulations, or a slow release apparatus will often be utilized
for continuous or long term administration. See, e.g., Langer
(1990) Science 249:1527-1533.
[0134] IL-D80 or IL-27, fragments thereof, and antibodies to it or
its fragments, antagonists, and agonists, may be administered
directly to the host to be treated or, depending on the size of the
compounds, it may be desirable to conjugate them to carrier
proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in
many conventional dosage formulations. While it is possible for the
active ingredient to be administered alone, it is preferable to
present it as a pharmaceutical formulation. Formulations typically
comprise at least one active ingredient, as defined above, together
with one or more acceptable carriers thereof. Each carrier should
be both pharmaceutically and physiologically acceptable in the
sense of being compatible with the other ingredients and not
injurious to the patient. Formulations include those suitable for
oral, rectal, nasal, topical, or parenteral (including
subcutaneous, intramuscular, intravenous and intradermal)
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any methods well known in
the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman
and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,
Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.
(1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993)
Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, New
York; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms:
Tablets, Dekker, New York; and Lieberman, et al. (eds. 1990)
Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York.
The therapy of this invention may be combined with or used in
association with other agents, e.g., other cytokines, including
IL-12, or its antagonists.
[0135] Both naturally occurring and recombinant forms of the IL-D80
or IL-27s of this invention are particularly useful in kits and
assay methods which are capable of screening compounds for binding
activity to the proteins. Several methods of automating assays have
been developed in recent years so as to permit screening of tens of
thousands of compounds in a short period. See, e.g., Fodor, et al.
(1991) Science 251:767-773, which describes means for testing of
binding affinity by a plurality of defined polymers synthesized on
a solid substrate. The development of suitable assays can be
greatly facilitated by the availability of large amounts of
purified, soluble IL-D80 or IL-27 as provided by this
invention.
[0136] Other methods can be used to determine the critical residues
in IL-D80 or IL-27 receptor interactions. Mutational analysis can
be performed, e.g., see Somoza, et al. (1993) J. Exptl. Med.
178:549-558, to determine specific residues critical in the
interaction and/or signaling. PHD (Rost and Sander (1994) Proteins
19:55-72) and DSC (King and Sternberg (1996) Protein Sci.
5:2298-2310) can provide secondary structure predictions of
.alpha.-helix (H), .beta.-strand (E), or coil (L). Helices A and D
are most important in receptor interaction, with the D helix the
more important region. Boundaries for the various helices are
indicated above. Surface exposed residues would affect receptor
binding, while embedded residues would affect general
structure.
[0137] For example, antagonists can normally be found once the
antigen has been structurally defined, e.g., by tertiary structure
data. Testing of potential interacting analogs is now possible upon
the development of highly automated assay methods using a purified
IL-D80 or IL-27. In particular, new agonists and antagonists will
be discovered by using screening techniques described herein. Of
particular importance are compounds found to have a combined
binding affinity for a spectrum of IL-D80 or IL-27 molecules, e.g.,
compounds which can serve as antagonists for species variants of
IL-D80 or IL-27.
[0138] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing an IL-D80 or IL-27. Cells may
be isolated which express an IL-D80 or IL-27 in isolation from
other molecules. Such cells, either in viable or fixed form, can be
used for standard binding partner binding assays. See also, Parce,
et al. (1989) Science 246:243-247; and Owicki, et al. (1990) Proc.
Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitive methods
to detect cellular responses.
[0139] Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to an IL-D80 or IL-27 and is described in
detail in Geysen, European Patent Application 84/03564, published
on Sep. 13, 1984. First, large numbers of different small peptide
test compounds are synthesized on a solid substrate, e.g., plastic
pins or some other appropriate surface, see Fodor, et al. (1991).
Then all the pins are reacted with solubilized, unpurified or
solubilized, purified IL-D80 or IL-27, and washed. The next step
involves detecting bound IL-D80 or IL-27.
[0140] Rational drug design may also be based upon structural
studies of the molecular shapes of the IL-D80 or IL-27 and other
effectors or analogs. Effectors may be other proteins which mediate
other functions in response to binding, or other proteins which
normally interact with IL-D80 or IL-27, e.g., a receptor. One means
for determining which sites interact with specific other proteins
is a physical structure determination, e.g., x-ray crystallography
or 2 dimensional NMR techniques. These will provide guidance as to
which amino acid residues form molecular contact regions, as
modeled, e.g., against other cytokine-receptor models. For a
detailed description of protein structural determination, see,
e.g., Blundell and Johnson (1976) Protein Crystallography, Academic
Press, New York.
[0141] IX. Kits
[0142] This invention also contemplates use of IL-D80 or IL-27
proteins, fragments thereof, peptides, and their fusion products in
a variety of diagnostic kits and methods for detecting the presence
of another IL-D80 or IL-27 or binding partner. Typically the kit
will have a compartment containing either a defined IL-D80 or IL-27
peptide or gene segment or a reagent which recognizes one or the
other, e.g., IL-D80 or IL-27 fragments or antibodies.
[0143] A kit for determining the binding affinity of a test
compound to an IL-D80 or IL-27 would typically comprise a test
compound; a labeled compound, for example a binding partner or
antibody having known binding affinity for IL-D80 or IL-27; a
source of IL-D80 or IL-27 (naturally occurring or recombinant); and
a means for separating bound from free labeled compound, such as a
solid phase for immobilizing the molecule. Once compounds are
screened, those having suitable binding affinity to the antigen can
be evaluated in suitable biological assays, as are well known in
the art, to determine whether they act as agonists or antagonists
to the IL-D80 or IL-27 signaling pathway. The availability of
recombinant IL-D80 or IL-27 polypeptides also provide well defined
standards for calibrating such assays.
[0144] A preferred kit for determining the concentration of, e.g.,
an IL-D80 or IL-27 in a sample would typically comprise a labeled
compound, e.g., binding partner or antibody, having known binding
affinity for the antigen, a source of cytokine (naturally occurring
or recombinant) and a means for separating the bound from free
labeled compound, e.g., a solid phase for immobilizing the IL-D80
or IL-27. Compartments containing reagents, and instructions, will
normally be provided.
[0145] Antibodies, including antigen binding fragments, specific
for the IL-D80 or IL-27 or fragments are useful in diagnostic
applications to detect the presence of elevated levels of IL-D80 or
IL-27 and/or its fragments. Such diagnostic assays can employ
lysates, live cells, fixed cells, immunofluorescence, cell
cultures, body fluids, and further can involve the detection of
antigens related to the antigen in serum, or the like. Diagnostic
assays may be homogeneous (without a separation step between free
reagent and antigen-binding partner complex) or heterogeneous (with
a separation step). Various commercial assays exist, such as
radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),
enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique
(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the
like. See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525;
Harlow and Lane (1980) Antibodies: A Laboratory Manual, CSH Press,
NY; and Coligan, et al. (eds. 1993) Current Protocols in
Immunology, Greene and Wiley, NY.
[0146] Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against an IL-D80 or IL-27, as such may be
diagnostic of various abnormal states. For example, overproduction
of IL-D80 or IL-27 may result in production of various
immunological reactions which may be diagnostic of abnormal
physiological states, particularly in proliferative cell conditions
such as cancer or abnormal activation or differentiation. Moreover,
the distribution pattern available provides information that the
cytokine is expressed in pancreatic islets, suggesting the
possibility that the cytokine may be involved in function of that
organ, e.g., in a diabetes relevant medical condition.
[0147] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the label, either labeled or unlabeled antibody or
binding partner, or labeled IL-D80 or IL-27 is provided. This is
usually in conjunction with other additives, such as buffers,
stabilizers, materials necessary for signal production such as
substrates for enzymes, and the like. Preferably, the kit will also
contain instructions for proper use and disposal of the contents
after use. Typically the kit has compartments for each useful
reagent. Desirably, the reagents are provided as a dry lyophilized
powder, where the reagents may be reconstituted in an aqueous
medium providing appropriate concentrations of reagents for
performing the assay.
[0148] Many of the aforementioned constituents of the drug
screening and the diagnostic assays may be used without
modification or may be modified in a variety of ways. For example,
labeling may be achieved by covalently or non-covalently joining a
moiety which directly or indirectly provides a detectable signal.
In any of these assays, the binding partner, test compound, IL-D80
or IL-27, or antibodies thereto can be labeled either directly or
indirectly. Possibilities for direct labeling include label groups:
radiolabels such as .sup.125I, enzymes (U.S. Pat. No. 3,645,090)
such as peroxidase and alkaline phosphatase, and fluorescent labels
(U.S. Pat. No. 3,940,475) capable of monitoring the change in
fluorescence intensity, wavelength shift, or fluorescence
polarization. Possibilities for indirect labeling include
biotinylation of one constituent followed by binding to avidin
coupled to one of the above label groups.
[0149] There are also numerous methods of separating the bound from
the free IL-D80 or IL-27, or alternatively the bound from the free
test compound. The IL-D80 or IL-27 can be immobilized on various
matrixes followed by washing. Suitable matrixes include plastic
such as an ELISA plate, filters, and beads. See, e.g., Coligan, et
al. (eds. 1993) Current Protocols in Immunology, Vol. 1, Chapter 2,
Greene and Wiley, NY. Other suitable separation techniques include,
without limitation, the fluorescein antibody magnetizable particle
method described in Rattle, et al. (1984) Clin. Chem. 30:1457-1461,
and the double antibody magnetic particle separation as described
in U.S. Pat. No. 4,659,678.
[0150] Methods for linking proteins or their fragments to the
various labels have been extensively reported in the literature and
do not require detailed discussion here. Many of the techniques
involve the use of activated carboxyl groups either through the use
of carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated olefin such
as maleimide, for linkage, or the like. Fusion proteins will also
find use in these applications.
[0151] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of an IL-D80 or IL-27. These sequences can be used as probes for
detecting levels of the IL-D80 or IL-27 message in samples from
patients suspected of having an abnormal condition, e.g.,
inflammatory or autoimmune. Since the cytokine may be a marker or
mediator for activation, it may be useful to determine the numbers
of activated cells to determine, e.g., when additional therapy may
be called for, e.g., in a preventative fashion before the effects
become and progress to significance. The preparation of both RNA
and DNA nucleotide sequences, the labeling of the sequences, and
the preferred size of the sequences has received ample description
and discussion in the literature. See, e.g., Langer-Safer, et al.
(1982) Proc. Nat'l. Acad. Sci. 79:4381-4385; Caskey (1987) Science
236:962-967; and Wilchek et al. (1988) Anal. Biochem. 171:1-32.
[0152] Diagnostic kits which also test for the qualitative or
quantitative expression of other molecules are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for combinations
of markers. See, e.g., Viallet, et al. (1989) Progress in Growth
Factor Res. 1:89-97. Other kits may be used to evaluate other cell
subsets.
[0153] X. Isolating an IL-D80 or IL-27 Receptor
[0154] Having isolated a ligand of a specific ligand-receptor
interaction, methods exist for isolating the receptor. See,
Gearing, et al. (1989) EMBO J. 8:3667-3676. For example, means to
label the IL-D80 or IL-27 cytokine without interfering with the
binding to its receptor can be determined. For example, an affinity
label can be fused to either the amino- or carboxyl-terminus of the
ligand. Such label may be a FLAG epitope tag, or, e.g., an Ig or Fc
domain. An expression library can be screened for specific binding
of the cytokine, e.g., by cell sorting, or other screening to
detect subpopulations which express such a binding component. See,
e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA 90:11267-11271;
and Liu, et al. (1994) J. Immunol. 152:1821-29. Alternatively, a
panning method may be used. See, e.g., Seed and Aruffo (1987) Proc.
Nat'l Acad. Sci. USA 84:3365-3369.
[0155] Protein cross-linking techniques with label can be applied
to isolate binding partners of the IL-D80 or IL-27 cytokine This
would allow identification of proteins which specifically interact
with the cytokine, e.g., in a ligand-receptor like manner. It has
been shown, as noted below, that the IL-27 composite cytokine binds
at least to an IL-12R-like subunit known as WSX-1/TCCR.
[0156] FACS analysis of detectably stained IL-D80, EBI3, and
WSX-1/TCCR molecules led to the finding that these molecules are
components in a receptor subunit/ligand complex. Specifically, the
composite cytokine of E-tagged hIL-D80 (hIL-D80E) and F-tagged
(Flag-tagged) hEBI3 (FhEBI3) binds to Baf3 cells expressing an
F-tagged version of WSX-1/TCCR, also referred to as hNR30. The
cells were stained using anti-E mAb and a PE-conjugated anti-mouse
Fab.sub.2 fragment. Co-immunoprecipitation experiments also
indicated that hIL-27 could be immunoprecipitated with R-tagged
(RGSH.sub.6-tagged) soluble WSX-1/TCCR (shNR3OR). Alternatively,
shNR3OR could be co-immunoprecipated in the presence of
hIL-D80E/FhEBI3 complex using anti-E or anti-F mAbs. These
experiments establish that WSX-1/TCCR is a receptor component of
the IL-27 composite cytokine Recent evidence shows that disrupting
the WSX-1/TCCR gene in mice results in lowered expression of
IFN.gamma., which is a critical cytokine in the mediation of
pro-inflammatory functions. These mice were unable to mount a Th1
response (See, e.g., Chen, et al. (2000) Nature 407:916-920.)
[0157] Experimental data indicates a possible role for the IL-27
composite cytokine in driving an inflammatory response. The
expression profile of EBI3 and IL-D80 overlaps in monocytes,
macrophages, and dendritic cells, indicating that the composite
cytokine is primarily produced by antigent presenting cells (APCs)
of the immune system. EBI3 has been shown to be upregulated in
colonic tissue of patients suffering from gut inflammation
disorders, e.g., ulcerative colitis, suggesting that the composite
cytokine may also be involved.
[0158] Taken together the above indicates a role for the composite
cytokine and its associated receptor subunit WSX-1/TCCR in
inflammatory responses. Therefore antagonizing the function of any
of the components in the receptor subunit:ligand complex should
have a beneficial effect in inflammatory diseases, e.g.,
inflammatory bowel disease, rheumatoid arthritis, etc.
EXAMPLES
[0159] I. General Methods
[0160] Many of the standard methods below are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning, A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor
Press, NY; Sambrook, et al. (1989) Molecular Cloning: A Laboratory
Manual (2d ed.) Vols. 1-3, CSH Press, NY; Ausubel, et al., Biology
Greene Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al.
(1987 and Supplements) Current Protocols in Molecular Biology
Wiley/Greene, NY; Innis, et al. (eds. 1990) PCR Protocols: A Guide
to Methods and Applications Academic Press, NY. Methods for protein
purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g., Ausubel, et
al. (1987 and periodic supplements); Deutscher (1990) "Guide to
Protein Purification," Methods in Enzymology vol. 182, and other
volumes in this series; Coligan, et al. (1995 and supplements)
Current Protocols in Protein Science John Wiley and Sons, New York,
NY; P. Matsudaira (ed. 1993) A Practical Guide to Protein and
Peptide Purification for Microsequencing, Academic Press, San
Diego, Calif.; and manufacturer's literature on use of protein
purification products, e.g., Pharmacia, Piscataway, N.J., or
Bio-Rad, Richmond, Calif. Combination with recombinant techniques
allow fusion to appropriate segments (epitope tags), e.g., to a
FLAG sequence or an equivalent which can be fused, e.g., via a
protease-removable sequence. See, e.g., Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, NY; and
Crowe, et al. (1992) QIAexpress: The High Level Expression &
Protein Purification System QUIAGEN, Inc., Chatsworth, Calif..
[0161] Standard immunological techniques are described, e.g., in
Hertzenberg, et al. (eds. 1996) Weir's Handbook of Experimental
Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; and Methods in Enzymology
vols. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and
163. Cytokine assays are described, e.g., in Thomson (ed. 1998) The
Cytokine Handbook (3d ed.) Academic Press, San Diego; Mire-Sluis
and Thorpe (1998) Cytokines Academic Press, San Diego; Metcalf and
Nicola (1995) The Hematopoietic Colony Stimulating Factors
Cambridge University Press; and Aggarwal and Gutterman (1991) Human
Cytokines Blackwell Pub.
[0162] Assays for vascular biological activities are well known in
the art. They will cover angiogenic and angiostatic activities in
tumor, or other tissues, e.g., arterial smooth muscle proliferation
(see, e.g., Koyoma, et al. (1996) Cell 87:1069-1078), monocyte
adhesion to vascular epithelium (see McEvoy, et al. (1997) J. Exp.
Med. 185:2069-2077), etc. See also Ross (1993) Nature 362:801-809;
Rekhter and Gordon (1995) Am. J. Pathol. 147:668-677; Thyberg, et
al. (1990) Atherosclerosis 10:966-990; and Gumbiner (1996) Cell
84:345-357.
[0163] Assays for neural cell biological activities are described,
e.g., in Wouterlood (ed. 1995) Neuroscience Protocols modules 10,
Elsevier; Methods in Neurosciences Academic Press; and Neuromethods
Humana Press, Totowa, N.J. Methodology of developmental systems is
described, e.g., in Meisami (ed.) Handbook of Human Growth and
Developmental Biology CRC Press; and Chrispeels (ed.) Molecular
Techniques and Approaches in Developmental Biology
Interscience.
[0164] FACS analyses are described in Melamed, et al. (1990) Flow
Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson,
et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y.
[0165] II. Cloning of Human IL-D80
[0166] The sequences of primate, e.g., human, genes are provided in
SEQ ID NO: 1, 3, 5, and 7. These sequences are derived from a
sequence database. These sequences allow preparation of PCR
primers, or probes, to determine cellular distribution of the gene.
These sequences allow isolation of genomic DNA which encode the
message.
[0167] Using the probe or PCR primers, various tissues or cell
types are probed to determine cellular distribution. PCR products
are cloned using, e.g., a TA cloning kit (Invitrogen). The
resulting cDNA plasmids are sequenced from both termini on an
automated sequencer (Applied Biosystems).
[0168] A structural alignment of available IL-6 family cytokine
folds (CNTF, LIF, IL-6, OSM and GCSF) from FSSP (see, e.g., Holm
and Sander (1998) Nucleic Acids Res. 26:316-319 was profile-aligned
to other sequences (including distant species variants of the
aforementioned cytokines, plus CT-1, GPA and viral IL-6's) with
Clustal X (see, e.g., Thompson, et al. (1997) Nucleic Acids Res.
25:4876-4882) with some manual adjustment. A weighted profile (see,
e.g., Thompson, et al. (1994) Nucleic Acids Res. 22:4673-4680) of
the most conserved region of the fold, the C-terminal D-helix
segment, a .about.40 amino acid block, was created. Fast scans of
sequence databases on a Bioccelerator machine (Compugen, Tel Aviv,
Israel) with the Profilesearch program (Gribskov et al., 1987)
identified human EST AI085007, mouse EST AA266872 and
eventually,the identification of a novel hemopoietic cytokine The
cytokine was initially referred to as IL-D80, but is also known as
p28 according to its apparent molecular mass as determined by
SDS-PAGE.
[0169] III. Cellular Expression of IL-D8 or IL-27
[0170] An appropriate probe or primers specific for cDNA encoding
primate IL-D80 or IL-27 are prepared. Typically, the probe is
labeled, e.g., by random priming.
[0171] Southern Analysis: DNA (5 .mu.g) from a primary amplified
cDNA library was digested with appropriate restriction enzymes to
release the inserts, run on a 1% agarose gel and transferred to a
nylon membrane (Schleicher and Schuell, Keene, N.H.).
[0172] Samples for human mRNA isolation may include: peripheral
blood mononuclear cells (monocytes, T cells, NK cells,
granulocytes, B cells), resting (T100); peripheral blood
mononuclear cells, activated with anti-CD3 for 2, 6, 12 h pooled
(T101); T cell, TH0 clone Mot 72, resting (T102); T cell, TH0 clone
Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled
(T103); T cell, TH0 clone Mot 72, anergic treated with specific
peptide for 2, 7, 12 h pooled (T104); T cell, TH1 clone HY06,
resting (T107); T cell, TH1 clone HY06, activated with anti-CD28
and anti-CD3 for 3, 6, 12 h 2 5 pooled (T108); T cell, TH1 clone
HY06, anergic treated with specific peptide for 2, 6, 12 h pooled
(T109); T cell, TH2 clone HY935, resting (T110); T cell, TH2 clone
HY935, activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled
(T111); T cell tumor lines Jurkat and Hut78, resting (T117); T cell
clones, pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69,
resting (T118); T cell random .gamma..delta.T cell clones, resting
(T119); CD28- T cell clone; Splenocytes, resting (B100);
Splenocytes, activated with anti-CD40 and IL-4 (B101); B cell EBV
lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting
(B102); B cell line JY, activated with PMA and ionomycin for 1, 6 h
pooled (B103); NK 20 clones pooled, resting (K100); NK 20 clones
pooled, activated with PMA and ionomycin for 6 h (K101); NKL clone,
derived from peripheral blood of LGL leukemia patient, IL-2 treated
(K106); hematopoietic precursor line TF1, activated with PMA and
ionomycin for 1, 6 h pooled (C100); U937 premonocytic line, resting
(M100); U937 premonocytic line, activated with PMA and ionomycin
for 1, 6 h pooled (M101);
[0173] elutriated monocytes, activated with LPS, IFN.gamma.,
anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102); elutriated
monocytes, activated with LPS, IFN.gamma., IL-10 for 1, 2, 6, 12,
24 h pooled (M103); elutriated monocytes, activated with LPS,
IFN.gamma., anti-IL-10 for 4, 16 h pooled (M106); elutriated
monocytes, activated with LPS, IFN.gamma., IL-10 for 4, 16 h pooled
(M107); elutriated monocytes, activated LPS for 1 h (M108);
elutriated monocytes, activated LPS for 6 h (M109); DC 70% CD1a+,
from CD34+GM-CSF, TNF.quadrature. 12 days, resting (D101); DC 70%
CD1a+, from CD34+ GM-CSF, TNF.quadrature. 12 days, activated with
PMA and ionomycin for 1 hr (D102); DC 70% CD1a+, from CD34+GM-CSF,
TNF.quadrature. 12 days, activated with PMA and ionomycin for 6 hr
(D103); DC 95% CD1a+, from CD34+ GM-CSF, TNF.quadrature. 12 days
FACS sorted, activated with PMA and ionomycin for 1, 6 h pooled
(D104); DC 95% CD14+, ex CD34+GM-CSF, TNF.quadrature. 12 days FACS
sorted, activated with PMA and ionomycin 1, 6 hr pooled (D105); DC
CD1a+CD86+, from CD34+GM-CSF, TNF.alpha. 12 days FACS sorted,
activated with PMA and ionomycin for 1, 6 h pooled (D106); DC from
monocytes GM-CSF, IL-4 5 days, resting (D107); DC from monocytes
GM-CSF, IL-4 5 days, resting (D108); DC from monocytes GM-CSF, IL-4
5 days, activated LPS 4, 16 h pooled (D109); DC from monocytes
GM-CSF, IL-4 5 days, activated TNF.alpha., monocyte supe for 4, 16
h pooled (D110); epithelial cells, unstimulated; epithelial cells,
IL-1.beta. activated; lung fibroblast sarcoma line MRCS, activated
with PMA and ionomycin for 1, 6 h pooled (C101); kidney epithelial
carcinoma cell line CHA, activated with PMA and ionomycin for 1, 6
h pooled (C102).
[0174] A rodent counterpart, e.g., mouse, has been identified, and
its distributions will be similarly evaluated. Samples for mouse
mRNA isolation can include: resting mouse fibroblastic L cell line
(C200); Braf:ER (Braf fusion to estrogen receptor) transfected
cells, control (C201); Mel14+naive T cells from spleen, resting
(T209); Mel14+naive T cells from spleen, stimulated with
IFN.quadrature., IL-12, and anti IL-4 to polarize to TH1 cells,
exposed to IFN.gamma. and IL-4 for 6, 12, 24 h, pooled (T210);
Mel14+ naive T cells from spleen, stimulated with IL-4 and anti
IFN.gamma. to polarize to Th2 cells, exposed to IL-4 and anti
IFN.gamma. for 6, 13, 24 h, pooled (T211); T cells, TH1 polarized
(Mel14 bright, CD430 cells from spleen, polarized for 7 days with
IFN-.quadrature. and anti IL-4; T200); T cells, TH2 polarized
(Mel14 bright, CD4+ cells from spleen, polarized for 7 days with
IL-4 and anti-IFN-.quadrature.; T201); T cells, highly TH1
polarized 3x from transgenic Balb/C (see Openshaw, et al. (1995) J.
Exp. Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 24 h
pooled; T202); T cells, highly TH2 polarized 3x from transgenic
Balb/C (activated with anti-CD3 for 2, 6, 24 h pooled (T203); T
cells, highly TH1 polarized 3.times. from transgenic C57 bl/6
(activated with anti-CD3 for 2, 6, 24 h pooled; T212); T cells,
highly TH2 polarized 3.times. from transgenic C57 bl/6 (activated
with anti-CD3 for 2, 6, 24 h pooled; T213); T cells, highly TH1
polarized (naive CD4+ T cells from transgenic Balb/C, polarized
3.times. with IFN.quadrature., IL-12, and anti-IL-4; stimulated
with IGIF, IL-12, and anti IL-4 for 6, 12, 24 h, pooled); CD44-
CD25+pre T cells, sorted from thymus (T204); TH1 T cell clone D1.1,
resting for 3 weeks after last stimulation with antigen (T205); TH1
T cell clone D1.1, 10 .quadrature.g/ml ConA stimulated 15 h (T206);
TH2 T cell clone CDC35, resting for 3 weeks after last stimulation
with antigen (T207); TH2 T cell clone CDC35, 10 .quadrature.g/ml
ConA stimulated 15 h (T208); unstimulated B cell line CH12 (B201);
unstimulated mature B cell leukemia cell line A20 (B200);
unstimulated large B cells from spleen (B202); B spleen, resting
(D200); dendritic cells from bone marrow, resting (D201);
unstimulated bone marrow derived dendritic cells depleted with anti
B220, anti CD3, and anti Class II, cultured in GM-CSF and IL-4
(D202); bone marrow derived dendritic cells depleted with anti
B220, anti CD3, and anti Class II, cultured in GM-CSF and IL-4,
stimulated with anti CD40 for 1, 5 d, pooled (D203); monocyte cell
line RAW 264.7 activated with LPS 4 h (M200); bone-marrow
macrophages derived with GM and M-CSF (M201); bone-marrow
macrophages derived with GM-CSF, stimulated with LPS,
IFN.quadrature., and IL-10 for 24 h (M205); bone-marrow macrophages
derived with GM-CSF, stimulated with LPS, IFN.quadrature., and anti
IL-10 for 24 h (M206); peritoneal macrophages (M207); macrophage
cell line J774, resting (M202); macrophage cell line
J774+LPS+anti-IL-10 at 0.5, 1, 3, 6, 12 h pooled (M203); macrophage
cell line J774+LPS+IL-10 at 0.5, 1, 3, 5, 12 h pooled (M204);
unstimulated mast cell lines MC-9 and MCP-12 (M208); immortalized
endothelial cell line derived from brain microvascular endothelial
cells, unstimulated (E200); immortalized endothelial cell line
derived from brain microvascular endothelial cells, stimulated
overnight with TNF.alpha.(E201); immortalized endothelial cell line
derived from brain microvascular endothelial cells, stimulated
overnight with TNF.alpha. (E202); immortalized endothelial cell
line derived from brain microvascular endothelial cells, stimulated
overnight with TNF.alpha. and IL-10 (E203); total aorta from wt C57
bl/6 mouse; total aorta from 5 month ApoE KO mouse (X207); total
aorta from 12 month ApoE KO mouse (X207); wt thymus (O214); total
thymus, rag-1 (O208); total kidney, rag-1 (O209); total kidney, NZ
B/W mouse; and total heart, rag-1 (O202). High signal was detected
in the monocyte cell line RAW 264.7 activated with LPS 4 h (M200);
T cells, highly TH1 polarized 3.times. from transgenic C57 bl/6
(activated with anti-CD3 for 2, 6, 24 h pooled; T212); and T cells,
highly TH1 polarized (naive CD4+ T cells from transgenic Balb/C,
polarized 3.times. with IFN.gamma., IL-12, and anti-IL-4;
stimulated with IGIF, IL-12, and anti IL-4 for 6, 12, 24 h,
pooled).
[0175] IV. Chromosome Mapping of IL-D80
[0176] An isolated cDNA encoding the IL-D80 is used. Chromosome
mapping is a standard technique. See, e.g., BIOS Laboratories (New
Haven, Conn.) and methods for using a mouse somatic cell hybrid
panel with PCR. The human IL-D80 gene is located on chromosome
16p11.
[0177] V. Expression and Purification of IL-D80 or IL-27
Proteins
[0178] Multiple transfected cell lines are screened for one which
expresses the cytokine at a high level compared with other cells.
Various cell lines are screened and selected for their favorable
properties in handling. Natural IL-D80 can be isolated from natural
sources, or by expression from a transformed cell using an
appropriate expression vector. Purification of the expressed
protein is achieved by standard procedures, or may be combined with
engineered means for effective purification at high efficiency from
cell lysates or supernatants. FLAG or His.sub.6 segments can be
used for such purification features. Alternatively, affinity
chromatography may be used with specific antibodies, see below.
[0179] cDNAs encoding full length human and mouse IL-D80 were
cloned into the pCDM8-etag vector via HindIII-XhoI (h/mp28-E).
EBI3: human and mouse EBI3 were cloned into pME18S-Ig vector via
EcoRI/XhoI (h/mEBI3-Ig) and the mature portion of human EBI3 into
pF1agCMV-1 vector via HindIII-NotI (F-hEBI3). One chain fusions
EBI3/p28: HindIII-XbaI fragments were generated encoding the mature
part of human or mouse EBI3, followed by the synthetic linker
GSGSGGSGGSGSGKL and by the mature coding sequence of human or mouse
IL-D80 via HindIII-NotI. Fragments were inserted into pFLAG-CMV-1
(Sigma) using HindIII-NotI sites.
[0180] WSX-1/TCCR: the preprotrypsin leader peptide and the flagtag
encoding part of pF1agCMV-1 vector were deleted by PCR, instead an
RGSH.sub.6-tag was introduced via SalI/SmaI (pCMV-1-RGSH.sub.6);
the cDNA encoding the extracellular part of human WSX-1 was cloned
into this vector via HindIII-SalI (soluble hWSX-1-R). In general
restriction sites were introduced through the respectively used PCR
primers and cDNA was ampified using standard PCR protocols.
Proteins were produced via transient expression in HEK293T cells.
For experiments requiring pure proteins purification was performed
by affinity chromatography using the respective protein tags.
[0181] VI. Transient Transfection, Metabolic Labeling and
Immunoprecipitation.
[0182] 1.times.10.sup.6 HEK293T cells were transiently transfected
with a total amount of 5 .mu.g plasmid DNA (control vector,
expression vectors encoding h/m p28-E, F-hEBI3 and mEBI3-Ig, or
respective combinations). Cells were cultured for 24 hr after
transfection, then metabolically labeled for 16 hr with 50
.mu.Ci/ml Pro-mix L-[.sup.35S] in vitro cell labeling mix (Amersham
Pharmacia) in cysteine/methionine free MEM. Proteins were
precipitated from supernatants with either anti-Flag M2 agarose
(Sigma), with anti-etag mAb bound to protein G sepharose (Amersham
Pharmacia), or with protein A sepharose (Amersham Pharmacia).
[0183] VII. Retroviral Constructs
[0184] The mature part of human and mouse WSX-1 was cloned into pMX
vector via HindIII-NotI, then a sequence encoding the preprotrypsin
leader peptide fused to a flag epitope was cloned into the vector
in frame and 5' of WSX-1 via BamHI-HindIII (F-h/mWSX-1). Retrovirus
obtained by transfection of BOSC23 cells was used to infect
parental Ba/F3 cells and cell surface expression of the desired
proteins was monitored using a flag-PE-staining in FACS
analysis.
[0185] VIII. Isolation of Homologous IL-D80 Genes
[0186] The IL-D80 cDNA, or other species counterpart sequence, can
be used as a hybridization probe to screen a library from a desired
source, e.g., a primate cell cDNA library. Many different species
can be screened both for stringency necessary for easy
hybridization, and for presence using a probe. Appropriate
hybridization conditions will be used to select for clones
exhibiting specificity of cross hybridization.
[0187] Screening by hybridization using degenerate probes based
upon the peptide sequences will also allow isolation of appropriate
clones. Alternatively, use of appropriate primers for PCR screening
will yield enrichment of appropriate nucleic acid clones.
[0188] Similar methods are applicable to isolate either species,
polymorphic, or allelic variants. Species variants are isolated
using cross-species hybridization techniques based upon isolation
of a full length isolate or fragment from one species as a
probe.
[0189] Alternatively, antibodies raised against human IL-D80 or
IL-27 will be used to screen for cells which express cross-reactive
proteins from an appropriate, e.g., cDNA library. The purified
protein or defined peptides are useful for generating antibodies by
standard methods, as described above. Synthetic peptides or
purified protein are presented to an immune system to generate
monoclonal or polyclonal antibodies. See, e.g., Coligan (1991)
Current Protocols in Immunology Wiley/Greene; and Harlow and Lane
(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press.
The resulting antibodies are used for screening, purification, or
diagnosis, as described.
[0190] IX. Preparation of antibodies specific for IL-D80 or
IL-27
[0191] Synthetic peptides or purified protein are presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold
Spring Harbor Press. Polyclonal serum, or hybridomas may be
prepared. In appropriate situations, the binding reagent is either
labeled as described above, e.g., fluorescence or otherwise, or
immobilized to a substrate for panning methods. Immunoselection,
absorptions, and related techniques are available to prepare
selective reagents, e.g., exhibiting the desired spectrum of
selectivity for binding.
[0192] X. Generation and Analysis of Genetically Altered
Animals
[0193] Transgenic mice can be generated by standard methods. Such
animals are useful to determine the effects of deletion of the
gene, in specific tissues, or completely throughout the organism.
Such may provide interesting insight into development of the animal
or particular tissues in various stages. Moreover, the effect on
various responses to biological stress can be evaluated. See, e.g.,
Hogan, et al. (1995) Manipulating the Mouse Embryo: A Laboratory
Manual (2d ed.) Cold Spring Harbor Laboratory Press.
[0194] IX. Expression/Distribution of IL-27
[0195] cDNAs from various libraries or cultured macrophages and
dendritic cells were prepared as described (see, e.g., Bolin, et
al. (1997) J. Neurosci. 17:5493-5502) and used as templates for
quantitative PCR. 50 ng cDNA was analyzed for expression of human
and mouse p28 and EBI3 by the fluorogenic 5'-nuclease PCR assay
(see, e.g., Holland, et al. (1991) Proc. Natl. Acad. Sci.
88:7276-7280) using the ABI Prism 7700 Sequence Detection System
(Perkin-Elmer, Foster City, Calif.). Analysis of cDNA samples was
corrected for expression of 18S rRNA using a VIC labeled probe
(Perkin-Elmer) in multiplex reactions.
[0196] Analysis of a large panel of human and mouse cDNA libraries
by real time quantitative PCR showed that expression of IL-D80 and
EBI3 is highly restricted. Both mRNAs are primarily found in cells
of myeloid lineage in human as well as mouse. Highest levels of
human mRNA's were found in LPS activated monocytes and monocyte
derived dendritic cells (DCs). A very high level of hEBI3 mRNA but
not hp28, was seen in placenta. This observation is in agreement
with earlier reports of high levels of EBI3 protein in placental
syncytiotrophoblasts [Devergne, 1997 #3]. A similar pattern emerged
when we analyzed the expression profile of mouse IL-D80 and EBI3.
Although mEBI3 was also expressed in some T and B cell libraries,
highest levels of both mIL-D80 and mEBI3 was in activated
macrophages.
[0197] Since antigen presenting cells are also the primary source
of IL-12 (see, e.g., Macatonia, et al. (1995) J. Immunol.
154:5071-5079) we studied the kinetics of production of IL-12p35,
IL-12p40, IL-D80 and EBI3 by monocyte derived DCs stimulated with
LPS. Human monocytes were isolated from peripheral blood,
stimulated with GM-CSF and IL-4 for 7 days to obtain immature DCs.
Subsequently, these CD14+CD11c+DCs were activated by LPS for
various time intervals and mRNA levels of IL-12p35, IL-12p40,
IL-D80 and EBI3 were analyzed by real time quantitative PCR.
Despite substantial variations in the absolute amounts of PCR
product from donor to donor and from protein to protein, the
kinetics recorded were consistent and revealed subtle differences
between the four investigated proteins. After an initial lag phase,
message levels for IL-12p35 and IL-12p40 rapidly increased and
consistently peaked between 8 and 14 hours of LPS stimulation, then
dropped back to background level after 24 hours. The profiles for
the two subunits of IL-12 are essentially superimposable. A very
transient expression was also observed for IL-D80, although maximal
message levels were already found after 3-6 hours. Similar to
IL-12, mRNA levels for p28 declined to background levels after 24
hours. In contrast, EBI3 showed less transient expression although
its transcription was also rapidly induced as early as 3 h after
LPS stimulus. Reaching maximal EBI3 mRNA levels between 12 and 24
hours, after 72 hours EBI3 message in all three donors was still
above the unstimulated background levels.
[0198] X. Transient Transfection, Metabolic Labeling, and
Immunoprecipitation
[0199] Appropriate host cells were transiently transfected with
empty vectors or expression vectors encoding hIL-D80E (E=E-tagged)
and/or FhEBI3 (F=Flag-tagged). Cells were cultured to 24 hrs. and
then metabolically labeled for 16 hrs with 50 .mu.Ci/ml PRO-MIX
L-[.sup.35S] in vitro cell labeling mix (Amersham Pharmacia) in
cysteine/methionine free MEM cell culture media. Proteins were
precipitated from 300 mL supernatant with either the anti-His5 mAb
or anti-E or anti-F mAb. The IL-12R like subunit, WSX-1/TCCR, was
also detectably labeled with RGSH.sub.6-tag (shNR30R) and
immunoprecipated as above.
[0200] XI. 2D-PAGE
[0201] Purified labeled IL-27 composite cytokine or
IL-27-WSX-1/TCCR complex were run on a nonreducing 10% NUPAGE gel
in MES running buffer (Novex). Appropriate lanes were excised,
reduced in sample buffer containing DTT, laid horizontally on
two-well 10% gels, and run reduced in a second dimension. One gel
was silver stained (Daiichi) while the other was blotted to a PVDF
membrane and developed using appropriate mAbs. It was found that
hIL-80E could be co-immunoprecipitated with shNR30R in the presence
of FhEBI3 using the anti-His.sub.6 mAb. Alternatively, shNR30R
could be immunoprecipated in the presence of hIL-80E and FhEBI3
using the anti-E mAb or anti-F mAb.
[0202] XII. Biological effects of IL-27
[0203] A. Naive Human and Mouse T Cells
[0204] CD4+CD45RB.sup.high or CD4+CD45RB.sup.low T cell subsets
were purified from the spleen and mesenteric lymph nodes of >6
month old IL-10-/-C57/B6 N12 mice as described (Davidson et al.,
1998). Cells were fractionated into CD4+CD45RB.sup.high and
CD4+CD45RB.sup.low cell populations by two color sorting on a
FACSTAR plus (Becton Dickinson). All populations were >99% pure
upon reanalysis. CD4+CD45RB.sup.high or CD4+CD45RB.sup.1' were put
into a proliferation assay with plate bound anti-CD3 (145.2C11)
stimulation as described (Davidson et al., 1998). Additions to the
growth media included anti-IL-2 Mab (JES6-1A12) 100 .mu.g/ml, and
cytokines as indicated. Cells were incubated for 5 days in a
humidified chamber (37.degree. C., 5% CO.sub.2) with [.sup.3H]TdR
(Amersham) added at a final concentration of 1 .mu.Ci/well for the
last 24h of incubation.
[0205] Sorted mouse naive T cells (CD4+CD45RB.sup.high) and
memory/activated T cells (CD4+CD45RB.sup.low) were stimulated with
CD3 mAb for four days in the presence of anti-IL-12 antibody and
various amounts of mIL-27. Upon stimulation, naive T cells, but not
memory T cells, showed a strong proliferative response.
Proliferation was augmented by addition of IL-12 at saturating
levels, revealing synergy between IL-27 and IL-12 on unstimulated T
cells. IL-27 was able to act as a strong expansion factor for
anti-CD3, anti-CD28 activated naive T cells in the absence of
IL-12.
[0206] FACS purified CD45RA and CD45RO T cells (purity>99%) were
cultured at a density of 4.times.10.sup.4 cells/well in a 96-well
plate previously coated with anti-CD3 antibody at 10 .mu.g/ml and
soluble anti-CD28 at 1 .mu.g/ml with or without IL-26/EBI3.
Anti-hIL-2 Mab 17H12 and anti-hIL-2R Mab B-B10 (Diaclone) were
added at 10 .mu.g/ml where indicated. IL-27 was also able to induce
proliferation of FACS sorted human CD45RA naive T cells isolated
from peripheral blood mononuclear cells (PBMC). Similar to the
results with mouse naive T cells, IL-27 induced strong
proliferation of CD3/CD28 naive T cells in the presence of
anti-IL-2. This response was enhanced by the addition of IL-12. No
response was seen with IL-27 treated CD45RO memory cells.
[0207] Thus, IL-27 dependent proliferation can be enhanced by
costimulatory signals through either CD28 or the IL-12 receptors.
IL-27 induced proliferation is dependent on simultaneous
crosslinking of CD3/TCR, since no proliferation was observed in the
absence of CD3 activation (data not shown). The same maximal
proliferative response could be induced by stimulation with
conditioned medium of p28/EBI3 co-transfected cells (data not
shown). To compare the abilities of IL-27 and IL-12 to induce
proliferation of naive CD4+T cells, FACS sorted mouse
CD4+CD45Rbhigh T cells were pre-cultured with plate bound anti-CD3
mAb, and either IL-27 or IL-12 were titrated into the cultures.
IL-27 proved to be a much more potent proliferative stimulus for
these cells (FIG. 4C).
[0208] Thus, IL-27 dependent proliferation can be enhanced by
costimulatory signals through either CD28 or the IL-12 receptors.
IL-27 induced proliferation is dependent on simultaneous
crosslinking of CD3/TCR, since no proliferation was observed in the
absence of CD3 activation. The same maximal proliferative response
could be induced by stimulation with conditioned medium of IL-27
co-transfected cells. To compare the abilities of IL-27 and IL-12
to induce proliferation of naive CD4+T cells, FACS sorted mouse
CD4+CD45RB.sup.high T cells were pre-cultured with plate bound
anti-CD3 mAb, and either IL-27 or IL-12 were titrated into the
cultures. IL-27 proved to be a much more potent proliferative
stimulus for these cells.
[0209] B. Induction of IFN-.gamma.
[0210] The ability of human and mouse IL-27 to induce the
production of IFN.gamma. in the presence of a neutralizing
anti-IL-2 mAb, with costimulation via anti-CD3 or
anti-CD3/anti-CD28 and both in the absence and presence of IL-12
was measured. In this assay neither hIL-27 nor hIL-12 by itself
induced IFN.gamma. production in anti-CD3 or anti-CD3/anti-CD28
activated CD4.sup.+CD45RA T cells. IFN.gamma. production was only
observed in the presence of both cytokines indicating strong
synergy between IL-27 and IL-12.
[0211] Sorted mouse CD4.sup.+CD45RB.sup.high naive T cells were
stimulated for 4 days with anti-CD3 mAb alone or with anti-CD3
mAb/anti-CD28 mAb and saturating amounts of IL-27 and IL-12. In the
absence of anti-CD28 costimulation neither IL-27 nor IL-12 by
itself was capable of inducing substantial amounts of IFN.gamma..
However, the combination of IL-27 and IL-12 induced up to about 300
ng/ml of IFN.gamma.. With anti-CD3/anti-CD28 costimulation, IL-27
as well as IL-12 were capable of inducing IFN.gamma. production.
The combination of both factors led to an additive effect with
IFN.gamma. levels up to 550 ng/ml.
[0212] C. IL-27 Does not Drive Th2 Polarization of Naive T
Cells
[0213] Sorted mouse CD4.sup.+CD45RBhigh T cells were cultured with
plate bound anti-CD3 and anti-CD28 in the presence of IL-4 and
IL-27. Including IL-27 in the cultures led to a decreased IL-13
production both in the absence and presence of IL-4 . Thus, while
inducing a strong Th1 response, IL-27 does not appear to promote
Th2 polarization.
[0214] D. IL-27 Binds to WSX-1/TCCR
[0215] Because of the relationship between IL-27 and the IL-6/IL-12
family, the search for the signaling receptors was concentrated on
this family. Members of this family were introduced into BaF3 cells
and tested for binding to IL-27. Of the receptors tested only Ba/F3
cells expressing the orphan cytokine receptor WSX-1/TCCR (see,
e.g., Sprecher, et al. (1998) Biochem. Biophys, Res. Comm.
246:82-90; and Chen, et al. (2000) Nature 407:916-920) showed
binding to tagged IL-27. BaF3 cells infected with retroviral
constructs expressing either F-tagged human or mouse WSX-1 cDNA
(F-hWSX-1 or F-mWSX-1) showed cellular staining using anti-Flag
mAb. Cells expressing F-hWSX-1 were then incubated with either
hEBI3-Ig alone or with coexpressed hIL-D80-E and EBI3-Ig for tow
hours. Heterodimeric IL-D80/EBI3 bound to WSX-1 while EBI3-Ig
itself showed no detectable binding. Similarly, only the
combination of mIL-D80-E and mEBI3-Ig provided a detectable
interaction with mWSX-1-expressing BaF3 cells, whereas the two
individual proteins were not able to do so. Incubation of
independently expressed mIL-D80-E and mEBI3-Ig with F-mWSX-1
expressing BaF3 cells also led to cellular staining. Untransfected
control cells were not stained by IL-D80/EBI3, demonstrating the
specificity of the observed interactions.
[0216] These results were confirmed by co-immunoprecipitation
experiments using a soluble extracellular form of hWSX-1 with a
C-terminal RSGH.sub.6-tag (R). Proteins from supernatants of
transiently transfected HEK293T cells containing F-hEBI3 or
coexpressed hILD80-E/F-hEBI3 were immunoprecipitated using either
Flag M2-agarose, agarose, protein G sepharose-coupled anti-etag mAb
or protein G sepharose-coupled anti-H.sub.5 mAb. The primary
pricipitates were washed and then incubated with HEK293T cell
supernatants containing shWSX-1-R. Secondary precipitates were
seperated by SDS-PAGE and subjected to western blot. Precipitaited
proteins were visualized by ECL using antibodies against the
respective protein tags. Only when all three proteins were present
(hIL-D80-E, F-hEBI3 and shWSX-1-R), immunoprecipitation of one
protein brought down both other components independently of the
immunoprecipitating antibody used. The same co-immunoprecipitation
experiment using the respective mouse orthologues had similar
results.
[0217] To address the question if WSX-1 was sufficient to mediate
IL-27 signal transduction, proliferation of BaF3 cells expressing
human or mouse WSX-1 was tested. These cells proliferate in
response to IL-3 but did not proliferate in response to IL-27. Thus
WSX-1 appears to be required but not sufficient for IL-27 mediated
signal transduction. The identification of additional IL-27 signal
transducing receptor subunits is currently in progress.
[0218] All references cited herein are incorporated herein by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference in its entirety for all purposes.
[0219] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
Sequence CWU 1
1
1211213DNAHomo sapiens 1cactggccca cgctgaagat aggggacttg agttccagtc
ttccttctgc taccgaccgg 60ctttgtgacc ttgaacaaga cttcccctcc ctgattccat
cctcatgtca catctgaagc 120ctccaacttc tgtcactgag ctcaggattc
ccaggcaagc ccacggagtg ccccacaggg 180tcagagccgt aacaggactt
ggaaaataac ccgaaaattg ggctcagcct gttgctgctt 240cccttgctcc
tggttcaagc tggtgtctgg ggattcccaa ggcccccagg gaggccccag
300ctgagcctgc aggagctgcg gagggagttc acagtcagcc tgcatctcgc
caggaagctg 360ctctccgagg ttcggggcca ggcccaccgc tttgcggaat
ctcacctgcc aggagtgaac 420ctgtacctcc tgcccctggg agagcagctc
cctgatgttt ccctgacctt ccaggcctgg 480cgccgcctct ctgacccgga
gcgtctctgc ttcatctcca ccacgcttca gcccttccat 540gccccgctgg
gagggctggg gacccagggc cgctggacca acatggagag gatgcagctg
600tgggccatga ggctggacct ccgcgatctg cagcggcacc tccgcttcca
ggtgctggct 660gcaggattca acctcccgga ggaggaggag gaggaagagg
aggaggagga ggaggagagg 720aaggggctgc tcccaggggc actgggcagc
gccttacagg gcccggccca ggtgtcctgg 780ccccagctcc tctccaccta
ccgcctgctg cactccttgg agctcgtctt atctcgggcc 840gtgcgggagt
tgctgctgct gtccaaggct gggcactcag tctggccctt ggggttccca
900acattgagcc cccagccctg atcggtggct tcttagcccc ctgcccccca
ccctttagaa 960ctttaggact ggagtcttgg catcagggca gccttcgcat
catcagcctt ggacaaggga 1020gggctcttcc agccccctgc cccaggccct
acccagtaac tgaaagcccc tctggtcctc 1080gccagctatt tatttcttgg
atatttattt attgtttagg gagatgatgg tttatttatt 1140gtcttggggc
ccgatggtcc tcctcgggcc aagcccccat gctgggtgcc caataaagca
1200ctctcatcca aaa 12132242PRTHomo sapiens 2Gln Asp Leu Glu Asn Asn
Pro Lys Ile Gly Leu Ser Leu Leu Leu Leu1 5 10 15Pro Leu Leu Leu Val
Gln Ala Gly Val Trp Gly Phe Pro Arg Pro Pro 20 25 30Gly Arg Pro Gln
Leu Ser Leu Gln Glu Leu Arg Arg Glu Phe Thr Val 35 40 45Ser Leu His
Leu Ala Arg Lys Leu Leu Ser Glu Val Arg Gly Gln Ala 50 55 60His Arg
Phe Ala Glu Ser His Leu Pro Gly Val Asn Leu Tyr Leu Leu65 70 75
80Pro Leu Gly Glu Gln Leu Pro Asp Val Ser Leu Thr Phe Gln Ala Trp
85 90 95Arg Arg Leu Ser Asp Pro Glu Arg Leu Cys Phe Ile Ser Thr Thr
Leu 100 105 110Gln Pro Phe His Ala Pro Leu Gly Gly Leu Gly Thr Gln
Gly Arg Trp 115 120 125Thr Asn Met Glu Arg Met Gln Leu Trp Ala Met
Arg Leu Asp Leu Arg 130 135 140Asp Leu Gln Arg His Leu Arg Phe Gln
Val Leu Ala Ala Gly Phe Asn145 150 155 160Leu Pro Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu Glu Glu Glu Arg 165 170 175Lys Gly Leu Leu
Pro Gly Ala Leu Gly Ser Ala Leu Gln Gly Pro Ala 180 185 190Gln Val
Ser Trp Pro Gln Leu Leu Ser Thr Tyr Arg Leu Leu His Ser 195 200
205Leu Glu Leu Val Leu Ser Arg Ala Val Arg Glu Leu Leu Leu Leu Ser
210 215 220Lys Ala Gly His Ser Val Trp Pro Leu Gly Phe Pro Thr Leu
Ser Pro225 230 235 240Gln Pro31098DNAMus
musculusmisc_feature(1)..(1)unknown amino 3nccaagntgg tacgcctgca
ggtaccggtc cggaattccc gggtcgaccc acgcgtccgg 60ggccaggtga caggagacct
tggctggcga ggactggaca ggcaacctgg ccaggagcag 120gactaaacag
acaaatgaag agtgtagagg gaagaggctg agaaccgagg acagtcagag
180gaacggcaca ggggagctgg gctcagcctg ttgctgctac ccttgcttct
ggtacaagct 240ggttcctggg ggttcccaac agaccccctg agccttcaag
agctgcgcag ggaattcaca 300gtcagcctgt accttgccag gaagctgctc
tctgaggttc agggctatgt ccacagcttt 360gctgaatctc gattgccagg
agtgaacctg gacctcctgc ccctgggata ccatcttcct 420aatgtttccc
tgactttcca ggcatggcat cacctctctg actctgagag actctgcttc
480ctcgctacca cacttcggcc cttccttgcc atgctgggag ggctggggac
ccaggggacc 540tggaccaaca tcaagaggat gcagcaatgg agactctctc
tggttcttga tgtggccctg 600tgtgtctttc gctcacaggt gctggctgca
ggattcaaat gttcaaagga ggaggaggac 660aaggaggaag aggaagagga
ggaagaagaa gaaaagaagc tgcccctagg gcgtctgggt 720ggccccaatc
aggtgtcatc ccaagtgtcc tggccccagc tgctctatac ctaccagctc
780cttcactcca tggagcttgt cctgtctcgg gctgttcggg acctgctgct
gctgtccctg 840cccaggcgcc caggctcagc cttggagttc ctaacaccta
gcttcaagcc ctgatggagt 900gaccttccag ctccctccct cgcccgttaa
gactctaagg ctggagtctg gccaatcaca 960ggacaggctc tagctcgttt
gccttagacc aggcagggtt tcactagctc ccagccctga 1020cccaataatt
taaaagccct ccagtcctta ccagatattt atttcttgga tatttattta
1080tttttaagaa atggttta 10984231PRTMus musculus 4Gly Leu Ser Leu
Leu Leu Leu Pro Leu Leu Leu Val Gln Ala Gly Ser1 5 10 15Trp Gly Phe
Pro Thr Asp Pro Leu Ser Leu Gln Glu Leu Arg Arg Glu 20 25 30Phe Thr
Val Ser Leu Tyr Leu Ala Arg Lys Leu Leu Ser Glu Val Gln 35 40 45Gly
Tyr Val His Ser Phe Ala Glu Ser Arg Leu Pro Gly Val Asn Leu 50 55
60Asp Leu Leu Pro Leu Gly Tyr His Leu Pro Asn Val Ser Leu Thr Phe65
70 75 80Gln Ala Trp His His Leu Ser Asp Ser Glu Arg Leu Cys Phe Leu
Ala 85 90 95Thr Thr Leu Arg Pro Phe Leu Ala Met Leu Gly Gly Leu Gly
Thr Gln 100 105 110Gly Thr Trp Thr Asn Ile Lys Arg Met Gln Gln Trp
Arg Leu Ser Leu 115 120 125Val Leu Asp Val Ala Leu Cys Val Phe Arg
Ser Gln Val Leu Ala Ala 130 135 140Gly Phe Lys Cys Ser Lys Glu Glu
Glu Asp Lys Glu Glu Glu Glu Glu145 150 155 160Glu Glu Glu Glu Glu
Lys Lys Leu Pro Leu Gly Arg Leu Gly Gly Pro 165 170 175Asn Gln Val
Ser Ser Gln Val Ser Trp Pro Gln Leu Leu Tyr Thr Tyr 180 185 190Gln
Leu Leu His Ser Met Glu Leu Val Leu Ser Arg Ala Val Arg Asp 195 200
205Leu Leu Leu Leu Ser Leu Pro Arg Arg Pro Gly Ser Ala Leu Glu Phe
210 215 220Leu Thr Pro Ser Phe Lys Pro225 2305732DNAHomo sapiens
5atgggccaga cggcaggcga ccttggctgg cggctcagcc tgttgctgct tcccttgctc
60ctggttcaag ctggtgtctg gggattccca aggcccccag ggaggcccca gctgagcctg
120caggagctgc ggagggagtt cacagtcagc ctgcatctcg ccaggaagct
gctctccgag 180gttcggggcc aggcccaccg ctttgcggaa tctcacctgc
caggagtgaa cctgtacctc 240ctgcccctgg gagagcagct ccctgatgtt
tccctgacct tccaggcctg gcgccgcctc 300tctgacccgg agcgtctctg
cttcatctcc accacgcttc agcccttcca tgccccgctg 360ggagggctgg
ggacccaggg ccgctggacc aacatggaga ggatgcagct gtgggccatg
420aggctggacc tccgcgatct gcagcggcac ctccgcttcc aggtgctggc
tgcaggattc 480aacctcccgg aggaggagga ggaggaagag gaggaggagg
aggaggagag gaaggggctg 540ctcccagggg cactgggcag cgccttacag
ggcccggccc aggtgtcctg gccccagctc 600ctctccacct accgcctgct
gcactccttg gagctcgtct tatctcgggc cgtgcgggag 660ttgctgctgc
tgtccaaggc tgggcactca gtctggccct tggggttccc aacattgagc
720ccccagccct ga 7326243PRTHomo sapiens 6Met Gly Gln Thr Ala Gly
Asp Leu Gly Trp Arg Leu Ser Leu Leu Leu1 5 10 15Leu Pro Leu Leu Leu
Val Gln Ala Gly Val Trp Gly Phe Pro Arg Pro 20 25 30Pro Gly Arg Pro
Gln Leu Ser Leu Gln Glu Leu Arg Arg Glu Phe Thr 35 40 45Val Ser Leu
His Leu Ala Arg Lys Leu Leu Ser Glu Val Arg Gly Gln 50 55 60Ala His
Arg Phe Ala Glu Ser His Leu Pro Gly Val Asn Leu Tyr Leu65 70 75
80Leu Pro Leu Gly Glu Gln Leu Pro Asp Val Ser Leu Thr Phe Gln Ala
85 90 95Trp Arg Arg Leu Ser Asp Pro Glu Arg Leu Cys Phe Ile Ser Thr
Thr 100 105 110Leu Gln Pro Phe His Ala Pro Leu Gly Gly Leu Gly Thr
Gln Gly Arg 115 120 125Trp Thr Asn Met Glu Arg Met Gln Leu Trp Ala
Met Arg Leu Asp Leu 130 135 140Arg Asp Leu Gln Arg His Leu Arg Phe
Gln Val Leu Ala Ala Gly Phe145 150 155 160Asn Leu Pro Glu Glu Glu
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 165 170 175Arg Lys Gly Leu
Leu Pro Gly Ala Leu Gly Ser Ala Leu Gln Gly Pro 180 185 190Ala Gln
Val Ser Trp Pro Gln Leu Leu Ser Thr Tyr Arg Leu Leu His 195 200
205Ser Leu Glu Leu Val Leu Ser Arg Ala Val Arg Glu Leu Leu Leu Leu
210 215 220Ser Lys Ala Gly His Ser Val Trp Pro Leu Gly Phe Pro Thr
Leu Ser225 230 235 240Pro Gln Pro7991DNAMus musculus 7atgggccaga
cggcaggcga ccttggctgg cggctcagcc tgttgctgct acccttgctt 60ctggtacaag
ctggttcctg ggggttccca acagaccccc tgagccttca agagctgcgc
120agggaattca cagtcagcct gtaccttgcc aggaagctgc tctctgaggt
tcagggctat 180gtccacagct ttgctgaatc tcgattgcca ggagtgaacc
tggacctcct gcccctggga 240taccatcttc ccaatgtttc cctgactttc
caggcatggc atcacctctc tgactctgag 300agactctgct tcctcgctac
cacacttcgg cccttccctg ccatgctggg agggctgggg 360acccagggga
cctggaccag ctcagagagg gagcagctgt gggccatgag gctggatctc
420cgggacctgc acaggcacct ccgctttcag gtgctggctg caggattcaa
atgttcaaag 480gaggaggagg acaaggagga agaggaagag gaggaagaag
aagaaaagaa gctgccccta 540ggggctctgg gtggccccaa tcaggtgtca
tcccaagtgt cctggcccca gctgctctat 600acctaccagc tccttcactc
cctggagctt gtcctgtctc gggctgttcg ggacctgctg 660ctgctgtccc
tgcccaggcg cccaggctca gcctgggatt cctaacacct agcttcaagc
720cctatggagt gaccttccag ctccctccct cgcccgttaa gactctaagg
ctggagtctg 780gccaatcaca ggacaggctc tagctcgttt gccttagacc
aggcagggct tcactagctc 840ccagccctga cccaataatt taaaagccct
ccagtcctta ccagatattt atttcttgga 900tatttattta tttttaagaa
atggtttatt tattgtttca ctcttgagtt aggccaccat 960gctgggtgcc
taataaagcc atccagcccg g 9918234PRTMus musculus 8Met Gly Gln Thr Ala
Gly Asp Leu Gly Trp Arg Leu Ser Leu Leu Leu1 5 10 15Leu Pro Leu Leu
Leu Val Gln Ala Gly Ser Trp Gly Phe Pro Thr Asp 20 25 30Pro Leu Ser
Leu Gln Glu Leu Arg Arg Glu Phe Thr Val Ser Leu Tyr 35 40 45Leu Ala
Arg Lys Leu Leu Ser Glu Val Gln Gly Tyr Val His Ser Phe 50 55 60Ala
Glu Ser Arg Leu Pro Gly Val Asn Leu Asp Leu Leu Pro Leu Gly65 70 75
80Tyr His Leu Pro Asn Val Ser Leu Thr Phe Gln Ala Trp His His Leu
85 90 95Ser Asp Ser Glu Arg Leu Cys Phe Leu Ala Thr Thr Leu Arg Pro
Phe 100 105 110Pro Ala Met Leu Gly Gly Leu Gly Thr Gln Gly Thr Trp
Thr Ser Ser 115 120 125Glu Arg Glu Gln Leu Trp Ala Met Arg Leu Asp
Leu Arg Asp Leu His 130 135 140Arg His Leu Arg Phe Gln Val Leu Ala
Ala Gly Phe Lys Cys Ser Lys145 150 155 160Glu Glu Glu Asp Lys Glu
Glu Glu Glu Glu Glu Glu Glu Glu Glu Lys 165 170 175Lys Leu Pro Leu
Gly Ala Leu Gly Gly Pro Asn Gln Val Ser Ser Gln 180 185 190Val Ser
Trp Pro Gln Leu Leu Tyr Thr Tyr Gln Leu Leu His Ser Leu 195 200
205Glu Leu Val Leu Ser Arg Ala Val Arg Asp Leu Leu Leu Leu Ser Leu
210 215 220Pro Arg Arg Pro Gly Ser Ala Trp Asp Ser225
23091161DNAHomo sapiensCDS(14)..(700)mat_peptide(74)..()
9gaattccgca gcc atg acc ccg cag ctt ctc ctg gcc ctt gtc ctc tgg 49
Met Thr Pro Gln Leu Leu Leu Ala Leu Val Leu Trp -20 -15 -10gcc agc
tgc ccg ccc tgc agt gga agg aaa ggg ccc cca gca gct ctg 97Ala Ser
Cys Pro Pro Cys Ser Gly Arg Lys Gly Pro Pro Ala Ala Leu -5 -1 1
5aca ctg ccc cgg gtg caa tgc cga gcc tct cgg tac ccg atc gcc gtg
145Thr Leu Pro Arg Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val
10 15 20gat tgc tcc tgg acc ctg ccg cct gct cca aac tcc acc agc ccc
gtg 193Asp Cys Ser Trp Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro
Val25 30 35 40tcc ttc att gcc acg tac agg ctc ggc atg gct gcc cgg
ggc cac agc 241Ser Phe Ile Ala Thr Tyr Arg Leu Gly Met Ala Ala Arg
Gly His Ser 45 50 55tgg ccc tgc ctg cag cag acg cca acg tcc acc agc
tgc acc atc acg 289Trp Pro Cys Leu Gln Gln Thr Pro Thr Ser Thr Ser
Cys Thr Ile Thr 60 65 70gat gtc cag ctg ttc tcc atg gct ccc tac gtg
ctc aat gtc acc gcc 337Asp Val Gln Leu Phe Ser Met Ala Pro Tyr Val
Leu Asn Val Thr Ala 75 80 85gtc cac ccc tgg ggc tcc agc agc agc ttc
gtg cct ttc ata aca gag 385Val His Pro Trp Gly Ser Ser Ser Ser Phe
Val Pro Phe Ile Thr Glu 90 95 100cac atc atc aag ccc gac cct cca
gaa ggc gtg cgc cta agc ccc ctc 433His Ile Ile Lys Pro Asp Pro Pro
Glu Gly Val Arg Leu Ser Pro Leu105 110 115 120gct gag cgc cac gta
cag gtg cag tgg gag cct ccc ggg tcc tgg ccc 481Ala Glu Arg His Val
Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro 125 130 135ttc cca gag
atc ttc tca ctg aag tac tgg atc cgt tac aag cgt cag 529Phe Pro Glu
Ile Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln 140 145 150gga
gct gcg cgc ttc cac cgg gtg ggg ccc att gaa gcc acg tcc ttc 577Gly
Ala Ala Arg Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe 155 160
165atc ctc agg gct gtg cgg ccc cga gcc agg tac tac gtc caa gtg gcg
625Ile Leu Arg Ala Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala
170 175 180gct cag gac ctc aca gac tac ggg gaa ctg agt gac tgg agt
ctc ccc 673Ala Gln Asp Leu Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser
Leu Pro185 190 195 200gcc act gcc aca atg agc ctg ggc aag
tagcaagggc ttcccgctgc 720Ala Thr Ala Thr Met Ser Leu Gly Lys
205ctccagacag cacctgggtc ctcgccaccc taagccccgg gacacctgtt
ggagggcgga 780tgggatctgc ctagcctggg ctggagtcct tgctttgctg
ctgctgagct gccgggcaac 840ctcagatgac cgacttttcc ctttgagcct
cagtttctct agctgagaaa tggagatgta 900ctactctctc ctttaccttt
acctttacca cagtgcaggg ctgactgaac tgtcactgtg 960agatattttt
tattgtttaa ttagaaaaga attgttgttg ggctgggcgc agtggatcgc
1020acctgtaatc ccagtcactg ggaagccgac gtgggtgggt agcttgaggc
caggagctcg 1080aaaccagtcc gggccacaca gcaagacccc atctctaaaa
aattaatata aatataaaat 1140aaaaaaaaaa aaaaggaatt c 116110229PRTHomo
sapiens 10Met Thr Pro Gln Leu Leu Leu Ala Leu Val Leu Trp Ala Ser
Cys Pro-20 -15 -10 -5Pro Cys Ser Gly Arg Lys Gly Pro Pro Ala Ala
Leu Thr Leu Pro Arg -1 1 5 10Val Gln Cys Arg Ala Ser Arg Tyr Pro
Ile Ala Val Asp Cys Ser Trp 15 20 25Thr Leu Pro Pro Ala Pro Asn Ser
Thr Ser Pro Val Ser Phe Ile Ala 30 35 40Thr Tyr Arg Leu Gly Met Ala
Ala Arg Gly His Ser Trp Pro Cys Leu45 50 55 60Gln Gln Thr Pro Thr
Ser Thr Ser Cys Thr Ile Thr Asp Val Gln Leu 65 70 75Phe Ser Met Ala
Pro Tyr Val Leu Asn Val Thr Ala Val His Pro Trp 80 85 90Gly Ser Ser
Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile Lys 95 100 105Pro
Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu Arg His 110 115
120Val Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu
Ile125 130 135 140Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln
Gly Ala Ala Arg 145 150 155Phe His Arg Val Gly Pro Ile Glu Ala Thr
Ser Phe Ile Leu Arg Ala 160 165 170Val Arg Pro Arg Ala Arg Tyr Tyr
Val Gln Val Ala Ala Gln Asp Leu 175 180 185Thr Asp Tyr Gly Glu Leu
Ser Asp Trp Ser Leu Pro Ala Thr Ala Thr 190 195 200Met Ser Leu Gly
Lys205112628DNAHomo
sapiensCDS(112)..(2022)misc_feature(2433)..(2433)unknown amino
11gtgggttcgg cttcccgttg cgcctcgggg gctgtaccca gagctcgaag aggagcagcg
60cggcccgcac ccggcaaggc tgggccggac tcggggctcc cgagggacgc c atg cgg
117 Met Arg 1gga ggc agg ggc ggc cct ttc tgg ctg tgg ccg ctg ccc
aag ctg gcg 165Gly Gly Arg Gly Gly Pro Phe Trp Leu Trp Pro Leu Pro
Lys Leu Ala 5 10 15ctg ctg cct ctg ttg tgg gtg ctt ttc cag cgg acg
cgt ccc cag ggc 213Leu Leu Pro Leu Leu Trp Val Leu Phe Gln Arg Thr
Arg Pro Gln Gly 20 25 30agc gcc ggg cca ctg cag tgc tac gga gtt gga
ccc ttg ggc gac ttg 261Ser Ala Gly Pro
Leu Gln Cys Tyr Gly Val Gly Pro Leu Gly Asp Leu35 40 45 50aac tgc
tcg tgg gag cct ctt ggg gac ctg gga gcc ccc tcc gag tta 309Asn Cys
Ser Trp Glu Pro Leu Gly Asp Leu Gly Ala Pro Ser Glu Leu 55 60 65cac
ctc cag agc caa aag tac cgt tcc aac aaa acc cag act gtg gca 357His
Leu Gln Ser Gln Lys Tyr Arg Ser Asn Lys Thr Gln Thr Val Ala 70 75
80gtg gca gcc gga cgg agc tgg gtg gcc att cct cgg gaa cag ctc acc
405Val Ala Ala Gly Arg Ser Trp Val Ala Ile Pro Arg Glu Gln Leu Thr
85 90 95atg tct gac aaa ctc ctt gtc tgg ggc act aag gca ggc cag cct
ctc 453Met Ser Asp Lys Leu Leu Val Trp Gly Thr Lys Ala Gly Gln Pro
Leu 100 105 110tgg ccc ccc gtc ttc gtg aac cta gaa acc caa atg aag
cca aac gcc 501Trp Pro Pro Val Phe Val Asn Leu Glu Thr Gln Met Lys
Pro Asn Ala115 120 125 130ccc cgg ctg ggc cct gac gtg gac ttt tcc
gag gat gac ccc ctg gag 549Pro Arg Leu Gly Pro Asp Val Asp Phe Ser
Glu Asp Asp Pro Leu Glu 135 140 145gcc act gtc cat tgg gcc cca cct
aca tgg cca tct cat aaa gtt ctg 597Ala Thr Val His Trp Ala Pro Pro
Thr Trp Pro Ser His Lys Val Leu 150 155 160atc tgc cag ttc cac tac
cga aga tgt cag gag gcg gcc tgg acc ctg 645Ile Cys Gln Phe His Tyr
Arg Arg Cys Gln Glu Ala Ala Trp Thr Leu 165 170 175ctg gaa ccg gag
ctg aag acc ata ccc ctg acc cct gtt gag atc caa 693Leu Glu Pro Glu
Leu Lys Thr Ile Pro Leu Thr Pro Val Glu Ile Gln 180 185 190gat ttg
gag cta gcc act ggc tac aaa gtg tat ggc cgc tgc cgg atg 741Asp Leu
Glu Leu Ala Thr Gly Tyr Lys Val Tyr Gly Arg Cys Arg Met195 200 205
210gag aaa gaa gag gat ttg tgg ggc gag tgg agc ccc att ttg tcc ttc
789Glu Lys Glu Glu Asp Leu Trp Gly Glu Trp Ser Pro Ile Leu Ser Phe
215 220 225cag aca ccg cct tct gct cca aaa gat gtg tgg gta tca ggg
aac ctc 837Gln Thr Pro Pro Ser Ala Pro Lys Asp Val Trp Val Ser Gly
Asn Leu 230 235 240tgt ggg acg cct gga gga gag gaa cct ttg ctt cta
tgg aag gcc cca 885Cys Gly Thr Pro Gly Gly Glu Glu Pro Leu Leu Leu
Trp Lys Ala Pro 245 250 255ggg ccc tgt gtg cag gtg agc tac aaa gtc
tgg ttc tgg gtt gga ggt 933Gly Pro Cys Val Gln Val Ser Tyr Lys Val
Trp Phe Trp Val Gly Gly 260 265 270cgt gag ctg agt cca gaa gga att
acc tgc tgc tgc tcc cta att ccc 981Arg Glu Leu Ser Pro Glu Gly Ile
Thr Cys Cys Cys Ser Leu Ile Pro275 280 285 290agt ggg gcg gag tgg
gcc agg gtg tcc gct gtc aac gcc aca agc tgg 1029Ser Gly Ala Glu Trp
Ala Arg Val Ser Ala Val Asn Ala Thr Ser Trp 295 300 305gag cct ctc
acc aac ctc tct ttg gtc tgc ttg gat tca gcc tct gcc 1077Glu Pro Leu
Thr Asn Leu Ser Leu Val Cys Leu Asp Ser Ala Ser Ala 310 315 320ccc
cgt agc gtg gca gtc agc agc atc gct ggg agc acg gag cta ctg 1125Pro
Arg Ser Val Ala Val Ser Ser Ile Ala Gly Ser Thr Glu Leu Leu 325 330
335gtg acc tgg caa ccg ggg cct ggg gaa cca ctg gag cat gta gtg gac
1173Val Thr Trp Gln Pro Gly Pro Gly Glu Pro Leu Glu His Val Val Asp
340 345 350tgg gct cga gat ggg gac ccc ctg gag aaa ctc aac tgg gtc
cgg ctt 1221Trp Ala Arg Asp Gly Asp Pro Leu Glu Lys Leu Asn Trp Val
Arg Leu355 360 365 370ccc cct ggg aac ctc agt gct ctg tta cca ggg
aat ttc act gtc ggg 1269Pro Pro Gly Asn Leu Ser Ala Leu Leu Pro Gly
Asn Phe Thr Val Gly 375 380 385gtc ccc tat cga atc act gtg acc gca
gtc tct gct tca ggc ttg gcc 1317Val Pro Tyr Arg Ile Thr Val Thr Ala
Val Ser Ala Ser Gly Leu Ala 390 395 400tct gca tcc tcc gtc tgg ggg
ttc agg gag gaa tta gca ccc cta gtg 1365Ser Ala Ser Ser Val Trp Gly
Phe Arg Glu Glu Leu Ala Pro Leu Val 405 410 415ggg cca acg ctt tgg
cga ctc caa gat gcc cct cca ggg acc ccc gcc 1413Gly Pro Thr Leu Trp
Arg Leu Gln Asp Ala Pro Pro Gly Thr Pro Ala 420 425 430ata gcg tgg
gga gag gtc cca agg cac cag ctt cga ggc cac ctc acc 1461Ile Ala Trp
Gly Glu Val Pro Arg His Gln Leu Arg Gly His Leu Thr435 440 445
450cac tac acc ttg tgt gca cag agt gga acc agc ccc tcc gtc tgc atg
1509His Tyr Thr Leu Cys Ala Gln Ser Gly Thr Ser Pro Ser Val Cys Met
455 460 465aat gtg agt ggc aac aca cag agt gtc acc ctg cct gac ctt
cct tgg 1557Asn Val Ser Gly Asn Thr Gln Ser Val Thr Leu Pro Asp Leu
Pro Trp 470 475 480ggt ccc tgt gag ctg tgg gtg aca gca tct acc atc
gct gga cag ggc 1605Gly Pro Cys Glu Leu Trp Val Thr Ala Ser Thr Ile
Ala Gly Gln Gly 485 490 495cct cct ggt ccc atc ctc cgg ctt cat cta
cca gat aac acc ctg agg 1653Pro Pro Gly Pro Ile Leu Arg Leu His Leu
Pro Asp Asn Thr Leu Arg 500 505 510tgg aaa gtt ctg ccg ggc atc cta
ttc ttg tgg ggc ttg ttc ctg ttg 1701Trp Lys Val Leu Pro Gly Ile Leu
Phe Leu Trp Gly Leu Phe Leu Leu515 520 525 530ggg tgt ggc ctg agc
ctg gcc acc tct gga agg tgc tac cac cta agg 1749Gly Cys Gly Leu Ser
Leu Ala Thr Ser Gly Arg Cys Tyr His Leu Arg 535 540 545cac aaa gtg
ctg ccc cgc tgg gtc tgg gag aaa gtt cct gat cct gcc 1797His Lys Val
Leu Pro Arg Trp Val Trp Glu Lys Val Pro Asp Pro Ala 550 555 560aac
agc agt tca ggc cag ccc cac atg gag caa gta cct gag gcc cag 1845Asn
Ser Ser Ser Gly Gln Pro His Met Glu Gln Val Pro Glu Ala Gln 565 570
575ccc ctt ggg gac ttg ccc atc ctg gaa gtg gag gag atg gag ccc ccg
1893Pro Leu Gly Asp Leu Pro Ile Leu Glu Val Glu Glu Met Glu Pro Pro
580 585 590ccg gtt atg gag tcc tcc cag ccc gcc cag gcc acc gcc ccg
ctt gac 1941Pro Val Met Glu Ser Ser Gln Pro Ala Gln Ala Thr Ala Pro
Leu Asp595 600 605 610tct ggg tat gag aag cac ttc ctg ccc aca cct
gag gag ctg ggc ctt 1989Ser Gly Tyr Glu Lys His Phe Leu Pro Thr Pro
Glu Glu Leu Gly Leu 615 620 625ctg ggg ccc ccc agg cca cag gtt ctg
gcc tga accacacgtc tggctggggg 2042Leu Gly Pro Pro Arg Pro Gln Val
Leu Ala 630 635ctgccagcca ggctagaggg atgctcatgc aggttgcacc
ccagtcctgg attagccctc 2102ttgatggatg aagacactga ggactcagag
aggctgagtc acttacctga ggacacccag 2162ccaggcagag ctgggattga
aggaccccta tagagaaggg cttggccccc atggggaaga 2222cacggatgga
aggtggagca aaggaaaata catgaaattg agagtggcag ctgcctgcca
2282aaatctgttc cgctgtaaca gaactgaatt tggaccccag cacagtggct
cacgcctgta 2342atcccagcac tttggcaggc caaggtggaa ggatcactta
gagctaggag tttgagacca 2402gcctgggcaa tatagcaaga cccctcacta
naaaaataaa acatcaaaaa caaaaacaat 2462tagctgggca tgatggcaca
cacctgtagt ccgagccact tgggaggctg aggtgggagg 2522atcggttgag
cccaggagtt cgaagctgca gggacctctg attgcaccac tgcactccag
2582gctgggtaac agaatgagac cttatctcaa aaataaacaa actaat
262812636PRTHomo sapiens 12Met Arg Gly Gly Arg Gly Gly Pro Phe Trp
Leu Trp Pro Leu Pro Lys1 5 10 15Leu Ala Leu Leu Pro Leu Leu Trp Val
Leu Phe Gln Arg Thr Arg Pro 20 25 30Gln Gly Ser Ala Gly Pro Leu Gln
Cys Tyr Gly Val Gly Pro Leu Gly 35 40 45Asp Leu Asn Cys Ser Trp Glu
Pro Leu Gly Asp Leu Gly Ala Pro Ser 50 55 60Glu Leu His Leu Gln Ser
Gln Lys Tyr Arg Ser Asn Lys Thr Gln Thr65 70 75 80Val Ala Val Ala
Ala Gly Arg Ser Trp Val Ala Ile Pro Arg Glu Gln 85 90 95Leu Thr Met
Ser Asp Lys Leu Leu Val Trp Gly Thr Lys Ala Gly Gln 100 105 110Pro
Leu Trp Pro Pro Val Phe Val Asn Leu Glu Thr Gln Met Lys Pro 115 120
125Asn Ala Pro Arg Leu Gly Pro Asp Val Asp Phe Ser Glu Asp Asp Pro
130 135 140Leu Glu Ala Thr Val His Trp Ala Pro Pro Thr Trp Pro Ser
His Lys145 150 155 160Val Leu Ile Cys Gln Phe His Tyr Arg Arg Cys
Gln Glu Ala Ala Trp 165 170 175Thr Leu Leu Glu Pro Glu Leu Lys Thr
Ile Pro Leu Thr Pro Val Glu 180 185 190Ile Gln Asp Leu Glu Leu Ala
Thr Gly Tyr Lys Val Tyr Gly Arg Cys 195 200 205Arg Met Glu Lys Glu
Glu Asp Leu Trp Gly Glu Trp Ser Pro Ile Leu 210 215 220Ser Phe Gln
Thr Pro Pro Ser Ala Pro Lys Asp Val Trp Val Ser Gly225 230 235
240Asn Leu Cys Gly Thr Pro Gly Gly Glu Glu Pro Leu Leu Leu Trp Lys
245 250 255Ala Pro Gly Pro Cys Val Gln Val Ser Tyr Lys Val Trp Phe
Trp Val 260 265 270Gly Gly Arg Glu Leu Ser Pro Glu Gly Ile Thr Cys
Cys Cys Ser Leu 275 280 285Ile Pro Ser Gly Ala Glu Trp Ala Arg Val
Ser Ala Val Asn Ala Thr 290 295 300Ser Trp Glu Pro Leu Thr Asn Leu
Ser Leu Val Cys Leu Asp Ser Ala305 310 315 320Ser Ala Pro Arg Ser
Val Ala Val Ser Ser Ile Ala Gly Ser Thr Glu 325 330 335Leu Leu Val
Thr Trp Gln Pro Gly Pro Gly Glu Pro Leu Glu His Val 340 345 350Val
Asp Trp Ala Arg Asp Gly Asp Pro Leu Glu Lys Leu Asn Trp Val 355 360
365Arg Leu Pro Pro Gly Asn Leu Ser Ala Leu Leu Pro Gly Asn Phe Thr
370 375 380Val Gly Val Pro Tyr Arg Ile Thr Val Thr Ala Val Ser Ala
Ser Gly385 390 395 400Leu Ala Ser Ala Ser Ser Val Trp Gly Phe Arg
Glu Glu Leu Ala Pro 405 410 415Leu Val Gly Pro Thr Leu Trp Arg Leu
Gln Asp Ala Pro Pro Gly Thr 420 425 430Pro Ala Ile Ala Trp Gly Glu
Val Pro Arg His Gln Leu Arg Gly His 435 440 445Leu Thr His Tyr Thr
Leu Cys Ala Gln Ser Gly Thr Ser Pro Ser Val 450 455 460Cys Met Asn
Val Ser Gly Asn Thr Gln Ser Val Thr Leu Pro Asp Leu465 470 475
480Pro Trp Gly Pro Cys Glu Leu Trp Val Thr Ala Ser Thr Ile Ala Gly
485 490 495Gln Gly Pro Pro Gly Pro Ile Leu Arg Leu His Leu Pro Asp
Asn Thr 500 505 510Leu Arg Trp Lys Val Leu Pro Gly Ile Leu Phe Leu
Trp Gly Leu Phe 515 520 525Leu Leu Gly Cys Gly Leu Ser Leu Ala Thr
Ser Gly Arg Cys Tyr His 530 535 540Leu Arg His Lys Val Leu Pro Arg
Trp Val Trp Glu Lys Val Pro Asp545 550 555 560Pro Ala Asn Ser Ser
Ser Gly Gln Pro His Met Glu Gln Val Pro Glu 565 570 575Ala Gln Pro
Leu Gly Asp Leu Pro Ile Leu Glu Val Glu Glu Met Glu 580 585 590Pro
Pro Pro Val Met Glu Ser Ser Gln Pro Ala Gln Ala Thr Ala Pro 595 600
605Leu Asp Ser Gly Tyr Glu Lys His Phe Leu Pro Thr Pro Glu Glu Leu
610 615 620Gly Leu Leu Gly Pro Pro Arg Pro Gln Val Leu Ala625 630
635
* * * * *
References